The biological activities of three purified preparations of heat-stable enterotoxin (ST), elaborated by different strains of Escherichia coli and known to differ in their amino acid composition and molecular size, were compared in the rabbit ileum. The mechanisms of action and potencies of all three purified STs were similar and resembled those previously demonstrated for partially purified ST. They all increased electrical potential difference and short-circuit current, inhibited active Cl- absorption, increased cyclic guanosine 3',5'-monophosphate production, and stimulated particulate guanylate cyclase activity in ileal mucosa. Their molar potencies were also similar, the concentrations of toxin required for half-maximal response differing less than fourfold in short-circuit current response and twofold in guanylate cyclase activity. However, there were 10-fold differences in potency when activity was expressed in mouse units per milliliter. Thus, heterogeneity in the size of these three ST molecules is not reflected in a difference in their mechanisms of action or potencies.