The binding of 5-fluoro-2'-deoxyuridylate, methylenetetrahydrofolate, and thymidylate synthetase in a ternary complex results in enzyme inhibition and is a major component of 5-fluorouracil cytotoxicity in some cells. The amount of 5-fluoro-2'-deoxyuridylate bound to thymidylate synthetase in several human gastrointestinal tumor cell lines following 5-fluorouracil exposure was determined, using Sephadex G-25 chromatography and high-pressure chromatographic analysis. These data were compared with previously determined values for thymidylate synthetase levels in control cultures not exposed to 5-fluorouracil. In HuTu 80 cells, the amount of 5-fluoro-2'-deoxyuridylate bound to thymidylate synthetase represented 16% of the total amount of enzyme present in untreated cells. The values for 5-fluoro-2'-deoxyuridylate bound to thymidylate synthetase in the WIDR and HT 29 cell lines (57 and 46 fmoles/10(5) cells, respectively), however, exceed by 3- to 6-fold the total amount of this enzyme found in untreated cells. The presence of increased levels of thymidylate synthetase protein in these cell lines was confirmed by sodium dodecyl sulfate gel electrophoresis. Measurements of thymidylate synthetase levels following exposure of cells to cycloheximide demonstrated that thymidylate synthetase complexed to 5-fluoro-2'-deoxyuridylate has increased stability as compared with uncomplexed enzyme. The level of thymidylate synthetase (bound and free) present in WIDR cells was measured following removal of FUra from the media. After 48 hr, the level of bound enzyme had fallen from 53 to 14 fmoles/10(5) cells, whereas free enzyme, which was undetectable after a 24-hr exposure to FUra, returned to 60% of its level in untreated cells.