The molecular basis for the treatment of human herpesviruses with nucleoside drugs is the phosphorylation of these drugs by the viral-encoded thymidine kinases. In order to better understand the structural and enzymatic mechanisms by which herpesviral thymidine kinases recognize their substrates, photoaffinity labeling with [alpha-32P]5-azido-2'-deoxyuridine-5'-monophosphate and [ gamma-32P]8-azidoadenosine-5'-triphosphate was used to characterize the thymidine, thymidylate, and ATP active sites of the herpes simplex virus-1 (HSV-1) thymidine kinase. For this study, HSV-1 thymidine kinase and a site-specific mutant enzyme (C336Y, known to confer acyclovir resistance) were expressed in bacteria and purified by a rapid, two-step protocol. The specificity of photoaffinity labeling of these HSV-1 thymidine kinases was demonstrated by the ability of site-directed substrates such as thymidine, thymidylate, acyclovir, 5-bromovinyl-2'-deoxyuridine, and ATP to inhibit photoinsertion. Differences in inhibition patterns of photoaffinity labeling correlated with kinetic differences between the wild-type and C336Y HSV-1 thymidine kinases. Cumulative results suggest that the acyclovir-resistant cysteine 336 mutation primarily affects the ATP binding site; yet it also leads to alteration in the binding affinity of nucleoside drugs in the thymidine site. In this study, azidonucleotide photoaffinity analogs are shown to be effective tools for studying the active-site environment of HSV-1 thymidine kinase and related site-specific mutants.