The catabolism of 5-fluorouracil (FUra), which accounts for 90% of the elimination of this antimetabolite in vivo, has recently been characterized in freshly isolated rat hepatocytes in suspension using a highly specific high-performance liquid chromatographic methodology. The present study evaluates the effect of thymine and uracil, which are thought to be catabolized by the same enzymes as FUra, on the metabolism and transmembrane distribution of FUra in isolated rat hepatocytes. Following simulataneous exposure of cells for 5 min to 30 microM [6-3H]FUra and increasing concentrations of either thymine or uracil, dihydrofluorouracil (FUH2) levels decreased in a concentration-dependent manner, and the concentration determined for 50% inhibition of FUra catabolism was 8.0 +/- 0.3 (S.D.) and 67.8 +/- 15.6 microM for thymine and uracil, respectively. Analysis of intracellular and extracellular 3H from 1 min to 2 hr after simultaneous incubation of the hepatocytes with 30 microM FUra and thymine (or uracil) in a 1:7 molar ratio resulted in a decrease of intracellular and extracellular FUH2 and alpha-fluoro-beta-alanine (FBAL), while alpha-fluoro-beta-ureidopropionic acid (FUPA) was enhanced. Unmetabolized FUra (not detected in the absence of thymine or uracil) was detected intracellularly in the presence of thymine or uracil and was accompanied by the appearance of a novel metabolite, preliminarily identified as a glucuronide of the FUra base which reached intracellular levels of 44 +/- 9.76 and 27.45 +/- 1.35 microM in the presence of thymine or uracil, respectively, within 1 hr. This metabolite, which penetrates the cell membrane only slowly, accounted for approximately 60% of the intracellular 3H in the presence of 300 microM FUra and 2 mM thymine, whereas FUra catabolism was inhibited by more than 99% under these conditions. The formation of FUra anabolites was insignificant in the presence of thymine and uracil, and incorporation of FUra into RNA was not enhanced. The lack of anabolism of FUra in isolated hepatocytes exposed to either high initial concentrations of FUra or high intracellular FUra concentrations resulting from modulation (inhibition) of FUra catabolism is consistent with the clinical observation of minimal hepatotoxicity with FUra, despite exposure of the liver to high blood levels. These studies indicate that thymine is a more potent modulator of FUra catabolism in hepatocytes than is uracil. Further studies are needed to clarify the biological importance of the glucuronide of the base FUra which accumulates intracellularly as the concentration of FUra increases within the hepatocytes.