Wings Paul Sackey and Mark Cueto have been formally ruled out of England's RBS 6 Nations Championship opener against Scotland at Twickenham next week.
Wasps flier Sackey is battling to overcome a knee injury that also threatens to sideline him from the Italy game on February 10.
Sale Sharks star Cueto, meanwhile, is laid low by a calf muscle problem.
England head coach Brian Ashton has trimmed his squad from 33 to 29 ahead of the Scotland clash, with Bristol scrum-half Shaun Perry and Gloucester lock Alex Brown also missing out.
Perry - who made his Test debut during England's autumn series - and Brown will join the England Saxons squad preparing to meet Italy A in Exeter next week.
Perry has been fighting to shake off a rib injury suffered during Bristol's Guinness Premiership victory over Bath two days after Christmas.
Leicester scrum-half Harry Ellis is favourite to wear the England number nine shirt against Scotland, with Peter Richards providing bench cover.
Ashton said: "The medical opinion was that Paul and Mark would not be fit, and I wish them all the best for a speedy recovery.
"Shaun and Alex have just missed the cut. It was a very tough decision, and underlines the strong competition for places."
With Sackey and Cueto unavailable, it is anticipated that Iain Balshaw, Josh Lewsey and Jason Robinson will fill the back-three slots for Scotland's visit.
Rugby league import Andy Farrell remains on course to make his Test debut at inside centre, while Ashton has aldo retained Jonny Wilkinson in the squad.
Newcastle star Wilkinson is expected to make a first appearance for 12 weeks in the Falcons' Premiership game against Leicester on Saturday after recovering from kidney damage.
But Wilkinson is unlikely to be considered for Test duty until the latter half of England's RBS 6 Nations season.
England's 29-man squad for RBS 6 Nations opener against Scotland at Twickenham on February 3:
Iain Balshaw (Gloucester), Mike Catt (London Irish), Harry Ellis (Leicester Tigers), Andy Farrell (Saracens), Toby Flood (Newcastle), Shane Geraghty (London Irish), Josh Lewsey (Wasps), Jamie Noon (Newcastle), Peter Richards (Gloucester), Jason Robinson (Sale), Mathew Tait (Newcastle), Mike Tindall (Gloucester), Jonny Wilkinson (Newcastle); George Chuter (Leicester), Martin Corry (Leicester), Louis Deacon (Leicester), Perry Freshwater (Perpignan), Danny Grewcock (Bath), Magnus Lund (Sale), Lee Mears (Bath), Lewis Moody (Leicester), Tom Palmer (Wasps), Tim Payne (Wasps), Tom Rees (Wasps), Steve Thompson (Northampton), Phil Vickery (capt, Wasps), Dan Ward-Smith (Bristol), Julian White (Leicester), Joe Worsley (Wasps).
The RBS 6 Nations Championship 2015 was not poised to go down as one of the more "vintage" Championships, though there was great anticipation as the action headed into its final day. What transpired was something that nobody could foresee: "The Greatest Day in Rugby's Greatest Championship."