5-Trifluoromethyl-2'-deoxyuridine (F(3)Thd), its free base and nucleotide triphosphate derivative, along with the nucleotide monophosphate and nucleotide triphosphate of deoxythymidine (dThd), were investigated as inhibitors of HEp-2 cell deoxythymidine kinase (dTK) and herpes simplex virus type 1 (HSV-1)-induced dTK. 5-Trifluoromethyluracil did not inhibit cellular or viral dTK. F(3)dThd competitively inhibited phosphorylation of dThd by both the HEp-2 cell- and the HSV-1-induced dTK. The K(mapp) for dThd and the K(Iapp) for the alternate substrate, F(3)dThd, were 3.5 and 22.5 muM for the HEp-2 cell dTK and 63.5 and 71.0 muM for the HSV-1-induced dTK. dThd-5'-PPP at 10 muM inhibited HEp-2 cell- and HSV-1-induced dTK by 94 and 22%, respectively. In comparison, 10 muM F(3)dThD-5'-PPP inhibited HEp-2 cell- and HSV-1-induced dTK 95 and 15%, respectively. These data indicate that F(3)dThd-5'-PPP may mimic dThd-5'-PPP feedback regulation of cellular and viral dTK.