The predictive utility of several biochemical parameters of 5-fluorouracil (5-FUra) action was evaluated in four murine colonic adenocarcinomas: 5-FUra-sensitive Tumor 38 and 5-FUra-resistant Tumors 07/A, 51 and 06/A. Thymidylate synthetase (TS) was determined by a tritiated 5-fluoro-2'-deoxyuridylate (FdUMP)-binding assay. Bolus 5-FUra (80 mg/kg, i.p.) administrated caused in all tumors a rapid decrease in free TS levels. Only Tumor 38, however, showed inhibition of TS to undetectable (less than 0.05 pmol/g) levels, which lasted up to 6 hr after treatment; correction for dissociation of endogenous TS: FdUMP:folate ternary complex during the TS assay was required. Total TS (free enzyme plus ternary complex) was determined with experimental conditions that achieved quantitative recovery of free TS from ternary complex. By 48 hr after 5-FUra, Tumor 38 showed a decrease in total TS proportional to the estimated log kill/dose of 5-FUra; in contrast, the resistant tumors showed no such decrease from pretreatment levels. Assay of FdUMP showed that the free nucleotide was formed rapidly in all tumors in excess over available TS-binding sites. However, tumor sensitivity did not correlate with peak or residual FdUMP levels or with deoxyuridylate levels, which were low and remained so in all tumors. Tumor sensitivity to 5-FUra also could not be explained by the small differences among the tumors in total perchloric acid-soluble metabolites of 5-FUra or drug incorporation into RNA. We conclude from these data that levels of free TS in the tumor after 5-FUra treatment are predictive of chemotherapeutic response in these murine models of human colonic adenocarcinoma.