England lock Maro Itoje has been left out of the squad for Saturday's crunch Six Nations match away to Wales after failing to recover in time from a knee injury.
Itoje sustained medial ligament damage during England's thumping 32-20 win over holders Ireland in their opening game although there was hope he would return in time for the top-of-the-table clash at Cardiff's Principality Stadium.
"England head coach Eddie Jones has retained 25 players to continue their preparation ahead of Saturday's Guinness Six Nations match against Wales in Cardiff," said the RFU in a statement. "Maro Itoje will remain at PennyHill Park this week to continue with his rehabilitation."
Chris Ashton had already been ruled out of the game with a calf strain, while prop Mako Vunipola will miss the remainder of the competition with an ankle problem.
England lead the Six Nations standings on 10 points after two games, leading Wales, who also won their opening games, due to bonus points from their victories over Ireland and France.
Full England Squad:
Forwards: Luke Cowan-Dickie, Tom Curry, Ellis Genge, Jamie George, Nathan Hughes, George Kruis, Joe Launchbury, Courtney Lawes, Ben Moon, Brad Shields, Kyle Sinckler, Billy Vunipola, Harry Williams, Mark Wilson.
Backs: Joe Cokanasiga, Elliot Daly, Owen Farrell (captain), George Ford, Jonny May, Jack Nowell, Dan Robson, Henry Slade, Ben Te'o, Manu Tuilagi, Ben Youngs.