A 1,268-bp polynucleotide probe for heat-labile and heat-stable enterotoxins (LTh, STIa, STIb) was conjugated with horseradish peroxidase (HRP). The HRP-conjugated trivalent probe was applied to the detection of enterotoxigenic Escherichia coli (ETEC) by colony and stool hybridizations. The binding of the probe to its targets was assayed by the addition of HRP substrates hydrogen peroxide and luminol in the presence of an enhancer, and the chemiluminescence was recorded by exposure to X-ray film. Slot blot hybridization demonstrated that the HRP-conjugated trivalent probe specifically hybridized with the DNA isolated from ETEC strains. The trivalent probe also specifically identified bacterial colonies of ETEC that produced LTh, STIa, STIb, LTh-STIa, or LTh-STIb. Treatment of targets with sodium dodecyl sulfate and proteinase K remarkably reduced nonspecific hybridization to DNAs of non-ETEC strains. Furthermore, this probe was able to detect stool specimens seeded with 10(2) original ETEC cells per 5 mg of feces. These results suggest that the HRP-conjugated trivalent probe is a candidate for use in the clinical laboratory to detect ETEC.