A sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the low molecular weight heat-stable enterotoxin (STa) in culture supernatant fluids of enterotoxigenic Escherichia coli (ETEC). Competitive inhibition was observed between STa in solution and a glutaraldehyde-coupled STa-human serum albumin (HSA) conjugate bound to microtiter wells when antiserum raised against a glutaraldehyde-coupled STa-bovine serum albumin (BSA) conjugate was used as detecting antibody. No competition was observed with conjugates prepared using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or dimethyl suberimidate and antisera raised against each conjugate. A biotin/avidin system increased the sensitivity of the assay such that 133 pg/ml of purified STa can be detected in less than 4 h. The assay was used to detect and quantify STa in culture supernatant fluids from human, porcine, and bovine ETEC isolates. No cross-reactivity was observed with the heat-labile enterotoxin (LT) or the form of ST with biological activity only in piglets (STb). Results from the quantitative STa ELISA showed good correlation (0.87) with the suckling mouse bioassay and a previously described radioimmunoassay. The quantitative assay was modified to reduce the total incubation time to less than 2 h. The qualitative STa ELISA provides a rapid and sensitive assay for clinical isolates of ETEC and should facilitate epidemiological studies on the incidence of STa-producing ETEC.