Thymidylate synthase (TS) is responsible for the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate. One of the principal mechanisms of action of 5-fluorouracil (5-FU) is the inhibition of TS by formation of a ternary covalent complex consisting of TS-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate. We have developed a Western immunoblot assay using the monoclonal antibody TS 106 to measure ternary complex and free TS in intact human carcinoma cells following exposure to either 5-FU alone or 5-FU plus leucovorin. Lysates from cells treated with either 5-FU or 5-FU/leucovorin were resolved in 15% polyacrylamide gel, transferred onto nitrocellulose and immunoblotted using TS 106 antibody. Detection of positive bands was by a chromogenic substrate strain. Immunoblotting detected free TS at 36 kDa and TS in ternary complex at 38.5 kDa which were quantitated by densitometric scanning. This assay was able to detect a ternary complex from intact cells treated with 5-FU or 5-FU/leucovorin up to 96 h after drug removal. The ratio of complex to free TS was up to 2-fold greater in 5-FU/leucovorin-treated cells compared to those treated with 5-FU alone. This assay may be applied to measuring the formation and stability of ternary complex and free TS in patient tissue samples.