I have setup the IIS website in windows 2008 R2 64bits server to allow user to get the file from UNC path.

Setting:

Only Windows authentication enabled (NTLM only).

Path credentials : Application user (pass-through authentication) - User will provide the AD account/password to access the resources.

Problem: 

The websites works fine for request around 3000 request a log, but when i switch more application user to new website with ~30000 request  (only three clients) a day (peak hour from 09:00 to 18:00).  From IIS log, we found that there are 15 minutes access fail with 401.3 error (Unauthorized Due to ACL on Resource) every day.  No this problem found in windows 2003 server with Intergrated windows authentication enabled only.

Anyone have similar problem encountered, please help. thanks