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We use the www sub-domain to serve html, css, etc for our site. We also have a www1 sub-domain that routes traffic to our application server. So right now, ISA routes www traffic to ServerA and www1 traffic to ServerB.

Are we able to create a folder on ServerB, say /handlers, that would be served up on the www sub-domain? If so, how?

For example, would be served from ServerB instead of ServerA.

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Yes. It's possible on newer versions of ISA Server. What version are you running? – Chris S Jul 7 '10 at 18:42
We are using 2006 Enterprise. – Bjørn Jul 7 '10 at 18:45

The ISA Server 2004 Web Publish instruction include publishing based on the folder. I believe the process is the same time 2006. About half way down that page you can find "Publishing Web server folders on the Internal or perimeter network to one domain name"

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