The Same
I Have the same problem on my 27" iMac.
This problem started after I downloaded LION and installed it.
After some 30 minutes or something Audio (and video) goes away.
No Audio from videos on Safari (youtube, vimeo etc), although the videos still plays.
Playing local video through QuickTime or MPlayerX won't play at all. If I play them with VLC or Lunettes (VLC Mac Beta) the video plays, but no video.
If I try to play an MP3 with QuickTime, Vox or MPlayerX the programs freeze and do nothing. If I try to play the MP3 with VLC or Lunettes the progress indicator moves, but theres no sound.
I have iTunes on autostart and auto-open a radiostream.
This works allright all trough the day, I can play music allready in the iTunes library and stream radio for as long as I want.
BUT, if I close iTunes completely and then start it up again the audio is gone.
This happens with both the internal and the external speakers.
Nothing in the audio settings seems out of place or are greyed out, looks just like its supposed to do.
The Microphone won't work either.
Systemsounds works just fine. EDIT: They don't work, but if I go to Settings-Sound-Soundeffects I can play the sounds.
Perian has been Un and Re-Installed.
The only cure is (more cowbell) a reboot of my iMac.
I sure hope nothings broken and this is only some software issue that will be fixed soon.