5-fluorouracil forms classic (covalent, ternary) complexes consisting of thymidylate synthase, fluoro-deoxyuridine monophosphate, and 5,10-methylene tetrahydrofolate. Despite a high pharmacologic interest in the classic complexes formed in cells treated with fluorouracil anticancer agents, the in vivo stability of the complexes and the possible interference in complex formation by other coadministered compounds have not been adequately described. We visualized classic complexes unaccompanied by unbound thymidylate synthase, inferring complete enzymatic inhibition, in 5-fluorouracil-treated S. cerevisiae and cancer cells in vitro and in murine tumors in vivo treated with 5-fluorouracil. Classic complexes persisted 13 days in cancer cells after a pulse of 5-fluorouracil. Classic complexes were reduced to absent in cancer cells in which the older antifolates methotrexate and aminopterin, or the modern antifolates pemetrexed and tomudex, were coadministered with 5-fluorouracil. Classic complexes were, however, detected when an alternate drug, 5-fluorodeoxyuridine, was administered with methotrexate. We visualized classic complexes at fifteen minutes to seven days after an acute single dose of 5-fluorouracil in mouse tumor models, in tumors and normal tissues. Using the same assay, we detected unbound thymidylate synthase in untreated human tissues, supporting the future use of this assay in evaluating the most appropriate dose of fluoropyrimidine and coadministered agents in clinical settings.