Eighteen temperature-sensitive (ts) mutants of herpes simplex virus type 1 were studied with regard to their ability to synthesize virus-induced thymidine kinase (TK) at permissive (34°) and nonpermissive (39°) temperatures. Five mutants were TK– at both temperatures (TK– mutants), 10 were TK+ at both temperatures (TK+ mutants) and 3 showed reduced TK activity at 39° but not at 34° (TK± mutants). Two of the 3 TK± mutants were ts in the production of TK at 39° while the third mutant synthesized a ts TK at both temperatures. Polyacrylamide gel electrophoresis of extracts of both uninfected and TK-mutant-infected cells exhibited one major and one minor peak of TK activity, whereas extracts of wild-type (WT) virus-infected cells revealed at least one additional major peak of TK activity. Gel patterns of TK± mutants were similar to those of the WT virus in 34 and 39° extracts; however, a reduction in TK activity of the viral peaks was observed in