Structure-based drug design methods were used to search for novel inhibitors of herpes simplex virus type 1 (HSV-1) thymidine kinase and Mycobacterium tuberculosis thymidylate kinase. The method involved the use of crystal structure complexes to guide database searching for potential inhibitors. A number of weak inhibitors of HSV-2 were identified, one of which was found to inhibit HSV-1 TK and HSV-1 TK-deficient viral strains. Each compound tested against M. tuberculosis thymidylate kinase was found to have some activity. The best of these compounds was only 4.6-fold less potent than 3'-azido-3'-deoxythymidine-5'-monophosphate (AZTMP). This study demonstrates the utility of structure-based drug design methods in the search for novel enzyme inhibitors.