Thymidylate synthetase levels in five human gastrointestinal tumor cell lines (two colon, two colorectal, one stomach) were determined. Titration of the enzyme in cell cytosol using the active-site titrant, 5-fluoro-2'-deoxyuridine-5'-monophosphate, demonstrated a 20-fold variation in the level of this enzyme among the tumor lines. Titrations performed in the presence or absence of added methylenetetrahydrofolate gave the same values for enzyme content. The cytotoxicity of 5-fluorodeoxyuridine to these cell lines (expressed as EC50 values) varied from 0.44 nM for SW 403 cells to 16 nM for HuTu 80 cells, and in all cases was reversed by the addition of thymidine. The concentration of 5-fluorodeoxyuridine required for cytotoxicity correlated directly (r = 0.98) with the level of thymidylate synthetase in the particular cell line. An inverse correlation (r = -0.95) was observed between the concentration of methotrexate producing cytotoxicity in these cell lines and their thymidylate synthetase levels. The cells were found to contain similar levels of dihydrofolate reductase and to possess normal transport capability for methotrexate.