This is perhaps related to this question, but I have slightly more information.
I recently updated an ASP.Net application to .NET 3.5 after coding a few new pieces with Linq. Now my pages intermittently stop firing event handlers. I have it narrowed down to pages with Ajax on them, and I assume it's either the ScriptManager or the AjaxControlToolkit registration that is precipitating the problem.
The really screwy part is that pages seem to work fine about 2/3s of the time, and the controls lose their event handlers only 1/3 of the time. I wondered about some type of error occurring and the page not rendering correctly, but I cannot find evidence of this.
We were have exactly this issue and as you have said it was only happening some of the time and only on pages with Ajax requests. We found that disabling the HTTP Keep-Alive as suggested in the answer to Why does Internet Explorer not send HTTP post body on Ajax call after failure? worked.
This can be done in IIS7 by following the instructions in How to configure HTTP KEEP-ALIVE in IIS 7