Manchester United full-back Aaron Wan-Bissaka and Aston Villa centre-back Tyrone Mings have won their first call-ups to the England senior squad.
Wan-Bissaka, who moved to Old Trafford from Crystal Palace this summer, was part of the Under-21 squad in June's age-grade European Championships.
Mings, 26, has made an assured start to his career at Villa since joining, initially on loan, in January.
Defender Kyle Walker, winner of 48 caps, is left out.
Gareth Southgate's side are preparing for Euro 2020 qualifiers against Bulgaria and Kosovo on 7 and 10 September respectively.
Of the two new faces, the England head coach said: "Tyrone's an interesting case as he entered the game a bit later and hasn't played as much as a centre-back.
"He had a big injury at Bournemouth but I really like his leadership qualities.
"He's an outstanding athlete and I see a lot of potential and time for growth given the number of games he's played.
"Aaron adapted very well to a high-profile move with the intensity and pressure."
Mings says he thought his England call-up was a prank when he first found out.
"I got a text when I came in from training from England's player liaison and I text her back, thinking she was joking," he said.
"She then called me and said she wasn't joking and that's how I found out. It was only when I spoke to her on the phone I realised it was serious."
Chelsea playmaker Mason Mount and Liverpool winger Alex Oxlade-Chamberlain are also in the 25-man squad.
Oxlade-Chamberlain's last England appearance was in 1-1 friendly draw against Italy in March 2018, month before he damaged knee ligaments playing for his club.
Walker, 29, is a right-back but was deployed as a central defender during England's run to the semi-finals of the World Cup in 2018.
His omission comes as Wan-Bissaka is called up and Atletico Madrid's former Tottenham right-back Kieran Trippier is recalled aftereing dropped for the Nations League finals.
Leicester playmaker James Maddison could be in line to win his first cap.
Who is left out?
Tottenham pair Dele Alli and Eric Dier, Everton's Fabian Delph and Manchester City defender John Stones have all been left out with injury problems.
Burnley striker Ashley Barnes, who has scored more goals than any other Englishman apart from Manchester City's Raheem Sterling in the Premier League this season, has been overlooked.
The 29-year-old is eligible to play for Austria, who he represented at under-20 level, through his grandmother but has not been called up for their senior side after politicians questioned whethere was sufficiently Austrian to win a full cap.
England manager Gareth Southgate is looking to the future while taking a step back into the past with his squad for the Euro 2020 qualifiers against Bulgaria and Kosovo.
The big surprise is the exclusion of Manchester City's Kyle Walker to make way for a first call-up for Manchester United's summer signing Aaron Wan-Bissaka - although Kieran Trippier's inclusion is arguably more contentious than that of the Old Trafford new boy given his poor form at Spurs last season before his move to Atletico Madrid.
Wan-Bissaka, 21, is another of Southgate's under-21 brigade to graduate into the full squad, joining Chelsea's Mason Mount. Mount has been included in England's squad before and takes his place fully on merit this time following an outstanding start to the season at Chelsea, where he has been given his chance by manager Frank Lampard and has responded with top-class performances and goals against Leicester City and Norwich City.
Walker had a poor Uefa Nations League Finals but is unfortunate to be left out.
Aston Villa central defender Tyrone Mings, meanwhile, is rewarded for fine form under Dean Smith and takes advantage of John Stones' absence through injury with his first call-up while a name from England's past makes a surprise return as Liverpool's Alex Oxlade-Chamberlain, fit again after a year out with a serious knee injury is recalled. He has not played for England since March 2018.
Leicester City's James Maddison, also only 22, will be hoping to get his chance after a superb start to the season as Southgate has demonstrated a willingness to blood young players, as proved by the fact Borussia Dortmund's Jadon Sancho is now a fixture in the squad at just 19. West Ham United's Declan Rice, just a year older, is also established in Southgate's senior group.
Walker's omission will be a major talking point. England are well blessed at right-back when Liverpool's Trent Alexander-Arnold is added to Wan-Bissaka and Trippier - but it is a sign Southgate is ready to be ruthless and take the big decisions to maintain England's forward momentum as they move towards next summer's Euro 2020.