Mason Mount and Ben Chilwell are "gutted" to have to sit out England's Euro 2020 clash against the Czech Republic.
The pair went into self-isolation following interaction with Chelsea team-mate Billy Gilmour, who tested positive for coronavirus.
The Three Lions' preparations for Tuesday's final Group D match were thrown into chaos when it emerged that the pair had been in contact with the Scotland midfielder, who returned a positive Covid-19 test following Friday night's Group D match.
Mount and Chilwell will now miss the pool decider at Wembley and could also be unavailable for England's last-16 match, with qualification assured although their place in the group is to be decided.
Following confirmation from the Football Association over the decision, which was taken in consultation with Public Health England (PHE), both players posted a brief message on social media.
Midfielder Mount wrote on Twitter: "I'm absolutely gutted to have to miss tonight's game. I'll be cheering on the boys as usual and ensuring that I'm ready to go again when called upon. Come on @England!"
Defender Chilwell posted: "It's a tough one to take and I'm gutted but I want to wish the boys all the best tonight in the game, I'll be cheering you all on. I will make sure I am ready for when I can rejoin the squad. Lets go @England!"
The FA said both players will now isolate "up to and including next Monday (June 28)".
A statement from the governing body read: "The pair were confirmed overnight as close contacts of Scotland's Billy Gilmour after his positive test following last Friday's match.
"Chilwell and Mount will isolate and train individually in private areas at England's training base St. George's Park, with the rest of the squad returning there after tonight's fixture against Czech Republic at Wembley.
"We will continue to follow all Covid-19 protocols and the UEFA testing regime, while remaining in close contact with PHE.
"The entire England squad and staff had lateral flow tests on Monday and all were again negative, as was the case with Sunday's UEFA pre-match PCR tests.
"Further tests will be carried out as and when appropriate."
Scotland's squad have been under the PHE umbrella, having stayed in Darlington and used Middlesbrough's training ground.
However, no other members of the group have been required to self-isolate following Gilmour's positive test.
Former Czech goalkeeper Petr Cech, now technical and performance advisor at Chelsea, questioned the logic behind such a scenario.
Writing on Instagram, Cech said: "Please could someone help me understand how is it possible Mason Mount and Ben Chilwell – both with negative tests – have to isolate after they got in contact with Billy Gilmour.
"While the entire Scottish team who shared the dressing room, hotel, coach and plane while travelling, dinning room, meeting room, is allowed to play the game without any problem because they returned the required negative tests?
"What is the difference between them returning the same test results? It is incomprehensible."