Deoxythymidine kinase (TdR kinase) from rabbit kidney cells infected in vitro with herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) was further investigated. Enzyme activity induced by HSV-2 was more heat-labile and more sensitive to inhibition by deoxythymidine nucleotides than HSV-1 induced enzyme activity in both crude extracts and partially purified preparations. The thermostability of TdR kinase activity from cells coinfected with HSV-1 and HSV-2 was intermediate between enzymes induced separately. The difference in thermostability between the two enzyme activities was also observed in extracts from 1-β-D-arabinofuranosylcytosine (ara-C) treated infected cells. HSV-1 and HSV-2 induced enzymes also differed in their response to substrate concentration. Gel filtration with Sephadex G-100 revealed the presence of monomer, dimer and aggregated forms of HSV-1 induced enzyme. The molecular weights of the monomer and dimer were estimated to be 58,000 and 97,000, respectively. Such multiple forms were not observed with HSV-2; the molecular weight of the HSV-2 induced enzyme was estimated to be 45,000 to 60,000, depending upon the eluting buffers. HSV-1 induced TdR kinase activity showed a different electrophoretic activity pattern in polyacrylamide gel electrophoresis than did the enzyme induced by HSV-2.