Five heat-stable enterotoxins (STs) produced by enterotoxigenic Escherichia coli strains of porcine, bovine, and human host origins have been purified to apparent homogeneity. The STs with biological activity in suckling mice and piglets (STaS) contained 18 amino acid residues, 10 amino acids with a high proportion of acidic amino acids, and 6 half-cystines. The carboxy-terminal and amino-terminal residues of all STaS were tyrosine and asparagine, respectively. All five STa preparations were homogeneous by several criteria: (i) a single symmetrical peak on gel filtration, (ii) a single fluorescent band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (iii) single carboxyl-terminal and amino-terminal residues, and (iv) amino acid analysis data that indicated a stoichiometric relationship between the component amino acids. The isoelectric points of the five STaS ranged from 3.88 to 4.08. All five purified preparations were heat stable and not denatured by organic solvents, detergents, or treatment at pH 1, but were partially inactivated after incubation at pH 12.0. Biological activity was completely abolished by treatment with reducing and oxidizing agents, which suggested that one or more disulfide bonds play an important role in the mechanism of action of STaS. Antisera raised against strain 431 STa, produced by a porcine class II enterpathogen, neutralized homologous 431 STa as well as heterologous purified STa preparations. The antiserum was used as a reagent in a sensitive radioimmunoassay to detect suckling mouse-positive strains of enterotoxigenic E. coli.