Basically you run a script at the sql2000 machine. Then run the output on the sql2005/sql2008 machine.
If the accounts are windows integrated they will just need to be created. But if they are sql accounts, they need to preserve their 'SQL' SID in order to work with the database, since its the sid, not the account name that connects them to a database.
Furthermore you need the hashed password so your users can continue use their passwords.
If your application are bought, the vendor should be able to assist in such a migration, that is if the application has support for SQL 2008.
Also check the following link:
http://support.microsoft.com/kb/246133