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 μM for the HEp-2 cell dTK and 63.5 and 71.0 μM for the HSV-1-induced dTK. dThd-5′-PPP at 10 μM inhibited HEp-2 cell- and HSV-1-induced dTK by 94 and 22%, respectively. In comparison, 10 μM 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.