The kinetic properties and control mechanisms of 5-fluorouracil (5-FU), uracil, and thymine degradation by rat liver dihydropyrimidine dehydrogenase were studied in vitro. The calculated Michaelis constant (Km) for 5-FU was 3.49 +/- 0.41 (SE) microM, similar to those for uracil (2.26 +/- 0.28 microM) and for thymine (2.23 +/- 0.34 microM). However, the reduction of 5-FU appears to be most sensitive to the inhibitory effects of increased substrate concentration. The specific activities of dihydropyrimidine dehydrogenase (nmol/min/mg of protein) for 5-FU, uracil, and thymine were 0.82, 0.68, and 0.56, respectively. Uridine was found to be a potent noncompetitive inhibitor of pyrimidine base degradation in vitro, displaying an inhibition constant (Ki) for 5-FU of 0.71 microM. Total inhibition of 5-FU degradation occurred at a uridine concentration of 10 microM, whereas thymidine was found to be a much less potent noncompetitive inhibitor of pyrimidine base degradation (Ki 24 microM). This paper provides the first documentation of in vitro inhibition of dihydropyrimidine dehydrogenase activity by nucleosides. The concomitant utilization of uridine and 5-FU in clinical situations might prove useful by decreasing 5-FU catabolism to toxic metabolites as well as enhancing 5-FU cytotoxicity.