Both failed the mark set by Chairman of selectors David Graveney, which was they had to play in at least one four-day Championship game - neither has played since the tour of India.
"Neither Michael nor Steve are in contention," confirmed an ECB spokesman.
Both left the India tour early, Vaughan with a knee injury and Harmison before the third Test with a shin problem.
The squad to face Sri Lanka at Lord's will be named on Sunday, four days before the match begins.
Vaughan, who has returned to running and batting in the nets, lost his last chance to make the squad when he failed to make the starting line-up for Yorkshire's match with Sussex, while Harmison's county Durham have no Championship game this week.
Andrew Flintoff, who led England to a 1-1 series tie in India, is likely to continue as stand-in captain, although management are worried by the all-rounder's workload.
But Flintoff denied the job provided an extra burden.
"I got an opportunity in India which I enjoyed but to do so at the home of cricket would be fantastic," said Flintoff.
"I enjoy being involved in the game and it's been quite an easy side to captain - captaining your country can't be a burden."
Vaughan's replacement in the batting looks like being a choice between two players.
Paul Collingwood stood in for Vaughan in Pakistan and India, scoring a maiden Test century in the first Test in Nagpur.
And Alastair Cook, who hit a century on debut in the same match, is also pushing for a place in the line-up.
Graveney and fellow selectors Duncan Fletcher and Geoff Miller must also patch up the pace-bowling attack with James Anderson already out with a back injury.
Simon Jones, who has a scan on his injured left knee on Thursday, is highly unlikely to figure.