The injection of 5-fluorouracil to partial hepatectomized rat prevented the rise of activities of thymidylate synthase and thymidine kinase as well as DNA synthesis in 24-h regenerating liver in a dose-dependent manner. The extent of the decrease of thymidine kinase activity was more than that of thymidylate synthase level. The immunoblotting assay showed the existence of fluorodeoxyuridine monophosphate bound thymidylate synthase in 5-fluorouracil injected rat liver. These results indicate that the reduction of DNA synthesis in regenerating liver by 5-fluorouracil result from the inhibitions of the rise of activities of both thymidylate synthase and thymidine kinase, while the mechanisms in their inhibitions may be different.