The DNA polymerases and thymidine kinases induced by three drug-resistant mutants of herpes simplex virus type 1 (S1, Tr7, and B3) and their common parent strain, SC16, have been purified and their properties compared. No significant differences were seen in the affinities of the polymerases for TTP and dGTP, or for the triphosphates of 9-(2-hydroxyethyloxymethyl)guanine (acyclovir) or (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) (drugs used in their isolation). In contrast all three mutants induced abnormal thymidine kinases. Those induced by the acyclovir-resistant mutants, S1 and Tr7, showed reduced affinities for thymidine, acyclovir, and also BVdU. Thymidine kinase induced by the BVdU-resistant mutant B3 showed reduced affinity for BVdU, but its affinities for thymidine and acyclovir were similar to those of the wild type enzyme. Thus, it appears that these variants of herpes simplex virus express altered thymidine kinases with impaired ability to phosphorylate particular nucleoside analogue drugs and these characteristics probably account for the drug resistance of the viruses. This strategy for resistance is important as it may result in variants with undiminished pathogenicity.