To assess the mechanism of the anticancer effect of doxifluridine (5'-DFUR), a clinicopharmacological study was performed. So far, no comparative study has been reported between the thymidylate synthase (TS) inhibition rate and the 5-FU concentration in colorectal cancer after oral administration of 5'-DFUR. In 37 patients with colorectal cancer, 5'-DFUR was administered orally/preoperatively (800 mg/day x more than 4 days before operation and 300 mg on the day of operation). The mean total dosage was 6.9 g. Specimens of tumor and normal intestinal tissue were obtained 4.4 hours on average after final administration. The TS inhibition rate as well as the 5-FU concentration and the activity of pyrimidine nucleoside phosphorylase (PyNPase) were analyzed in both tissues. The PyNPase activity was significantly higher in the tumor tissue than in the normal tissue (137.9 +/- 10.8 vs. 31.0 +/- 4.7 micrograms FU/mg protein/hr, p < 0.0001). The 5-FU concentration was also significantly higher in the tumor tissue than in the normal tissue (101.3 +/- 30.6 vs. 23.2 +/- 5.5 ng/g, p = 0.024). The TS inhibition rate correlated with the 5-FU concentration in the tumor tissue (r = 0.527, p = 0.047). These findings suggest that the TS inhibition rate may be an index of the anticancer effect of 5'-DFUR in colorectal cancer.