Thymidylate synthase (TS; EC 2.1.1.45) is an important therapeutic target for fluoropyrimidine cytotoxic drugs that are widely used for the treatment of solid tumors. Using the monoclonal antibody TS 106, we have developed an ultrasensitive enzyme-linked immunoassay (ELISA) for the detection and quantitation of TS. Using a chemiluminescent ELISA technique, TS was detectable in serially diluted lysates from NCI H630 and HCT 116 human colon carcinoma cell lines. The ELISA assay was reliably able to detect activity down to a level of 30 attamol of TS protein above background (P2 = 0.016). The usable range of detection was from 0.03 to 500 fmol of enzyme. There was a close correlation between the optical density signal and the total TS enzyme between both cell lines (r2 = 0.96). The ELISA was used to measure TS in cytosolic extracts from human tumor samples, and it was able to quantitate TS levels using as little as 1-mg tumor biopsy samples. The mean total TS measured by ELISA in seven tumor samples from patients with breast cancer and sarcomas was 131 fmol/mg cytosolic protein (range 60-240) compared with a mean TS of 85 fmol/mg cytosolic protein (range 35-163) using the fluorodeoxyuridine monophosphate binding assay. While the TS levels were uniformly higher when measured by ELISA, there was close proportional agreement between both assays (r2 = 0.84). Thus, the chemiluminescent TS ELISA would appear to be an extremely sensitive and specific assay that may be used to quantitate TS in tumor tissue specimens.