Trifluridine (TFT) and a structurally related analogue, 5-fluoro-2′-deoxyuridine (FDU), were investigated for their efficacy in the topical treatment of experimental keratitis caused by thymidine kinase-positive (TK+) and thymidine kinase-deficient (TK−) herpes simplex virus type 1 (HSV-1) strains. Bromovinyldeoxyuridine (BVDU) was used as a reference compound. Both 0.2 % BVDU and 0.2 % TFT eyedrops produced a highly significant healing of TK+HSV-1 keratitis as compared to the placebo and 0.2 % FDU eyedrops (P≪0.005), whereas the latter compound did not differ from placebo eyedrops. In the treatment of TKHSV-1 keratitis, none of the drugs exhibited a beneficial healing effect, although the virus strain used was inhibitedinvitroby TFT and FDU at a very low concentration (0.02–0.04μg/mL).