We investigated the relationships between antitumor activity and the inhibition of Thymidylate Synthase (TS) activity after oral administration of 5-fluorouracil (FUra) and its derivatives, FT, UFT, HC-FU, PH-FU and 5'-DFUR, using mainly Sarcoma 180 as an experimental tumor model. The inhibition of TS activity in the tumor, after oral administration of these drugs to Sarcoma 180 bearing mice, reached the plateau phase shortly after drug administration. The inhibition of TS activity remained at the same level for over 24 hours and was dose dependent. UFT and FT showed a very high correlation between the inhibition of TS activity and their antitumor activity. However, such a correlation was not found for other FUra derivatives despite their high TS inhibiting values. The relationship between the tumor growth inhibition (TGI) and the TS inhibition (TSI) of these drugs, presented as a ratio of ED50 of TGI to ED50 of TSI in case of UFT and FT, was shown to be 0.81 and 1.18, respectively (i.e., nearly 1.00). However, the above ratio for FUra and HCFU was shown to be 2.49 and 3.88, respectively. These findings demonstrate that it is necessary to take into account other mechanisms besides TS inhibition of some fluorinated pyrimidines to explain their total antitumor activity.