Antiserum was raised against the purified heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli strain 431, a class II porcine enteropathogen. The antiserum was used to examine the antigenic determinants of STs produced by enterotoxigenic strains of different host origins and develop a sensitive radioimmunoassay specific for ST having biological activity in suckling mice and piglets (STA). The antiserum neutralized one effective dose of toxin at a dilution of 1:5,000 and neutralized approximately 40 microgram of toxin per ml of serum. In the radioimmunoassay, protein A-bearing staphylococci was used as the primary solid-phase adsorbent. The purified STs produced by a class I enteropathogen (strain 667) and by a bovine enterotoxigenic strain (B-41) exhibited patterns of competitive inhibition identical to those of homologous unlabeled strain 431 ST in the radioimmunoassay when specific antibody to strain 431 ST was used. The levels of ST in culture supernatants determined by the suckling mouse assay correlated with the concentrations of toxin measured by the radioimmunoassay. The antiserum was specific for STA produced by enterotoxigenic E. coli of porcine, bovine, and human origins and did not react with heat-labile enterotoxin or with ST that had biological activity in piglets but not in suckling mice (STB). These results suggest that STA molecules having different host origins share at least one antigenic determinant.