The effect of mutational loss of thymidine kinase (TK) on the sensitivity to alkylating agents was investigated in promyelocytic, HL-60, and T-lymphoblastoid, Molt-3, human leukemia cell lines. Although both cell lines exhibited approx. 1% residual TK activity, only HL-60 TK deficient cells had a decreased intracellular TTP pool, i.e., 20% of that of the wild-type. When treated with N-methyl-N'-nitronitrosoguanidine or ethyl methanesulfonate, HL-60 TK deficient cells showed significantly increased killing and mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus relative than did wild-type. Pretreatment of cells with O6-benzylguanine, an inhibitor of O6-alkylguanine-DNA alkyltransferase, partially abolished those differences. Molt-3 wild-type and TK deficient cells had similar cell survivals and HGPRT mutation frequencies following treatment with alkylating agents. These results indicate that TK deficiency, only when a concomitant decrease of TTP pool is detected, plays a pivotal role in the sensitivity to the cytotoxic and mutagenic effects of alkylating agents.