The effects of trifluorothymidine (5-trifluoromethyl-2'-deoxyuridine, F3dThd) on the replication of three mouse cell lines, LM929, Ltk- (and LM929 derivative devoid of cytosolic deoxythymidine [dThd] kinase activity), and Ltk- c139 (a Ltk- derivative which expresses herpes simplex virus type 1-specified dThd kinase subsequent to biochemical transformation with ultraviolet-irradiated herpes simplex virus type 1), have been investigated. Complete inhibition of Ltk- cell growth required a 10,000-fold higher concentration of F3dThd (1.0 mM) than was required to completely inhibit LM929 and Ltk- c139 cell growth. The plating efficiency of exponentially dividing Ltk- cells after exposure to F3dThd (10 microM) for 24 h was 63% as compared to 3% for exponentially dividing LM929 cells. Stationary LM929 cells (confluent cultures held for a 6-day period in serum-reduced medium) with reduced dThd kinase specific activity and deoxyribonucleic acid biosynthesis level exhibited a plating efficiency similar to that of exponentially dividing Ltk- cells after exposure to F3Thd (1.0 mM) for 24 h. In addition, treatment of exponentially dividing LM929 and Ltk- cells with F3dThd (10 microM) for 24 h resulted in approximately an 80% and 25% reduction in deoxyribonucleic acid biosynthesis, respectively. These data indicated a requirement for cytosolic dThd kinase in the expression of F3dThd-induced cytotoxicity. F3dThd was shown to be a linear competitive inhibitor with respect to dThd for affinity-purified LM929 cytosolic dThd kinase. The Km(app) for dThd and Ki(app) for F3dThd with the cytosolic dThd kinase were 2.4 and 3.8 microM, respectively.