Drupal 6 will not use updated htaccess 301 information
.htaccess shouldn't require any cache flushes, restarts of the server, etc. They should just take effect. 

If you want to check to make sure that your editing the right file (and your not on a production server) you can always throw and invalid line in your .htaccess file. If it throws back a 500 error, your editing the right file and something is wrong somewhere in it, if it doesn't your're working on the wrong file.