Enterotoxin, a diarrhea-inducing protein elaborated by pathogenic Escherichia coli strains, was isolated from the supernate of fermenter cultures of E. coli strain P263, a porcine enteropathogen. Purification involved chromatography and preparative isotachophoresis. The resulting product appeared to be pure according to immunoelectrophoretic, disc electrophoretic, ultracentrifugal, and immunologic criteria. The enterotoxin had an apparent molecular weight of 102,000 daltons, and its isoelectric point was 6.90. The isolated product was active in inducing experimental diarrhea in adult rabbits and piglets. In small dosage it also elicited a drastic increase in adenylate cyclase activity in broken-cell preparations of cat heart tissue. The enterotoxin activity was acid labile and was destroyed by heat (65 C for 30 min). It is suggested that the heat-stable enterotoxin was derived from heat-labile enterotoxin by complexing with endotoxin or with capsular material in the culture supernatant. The antigenic relations between the heat-labile enterotoxins of enteropathogenic E. coli strains of different serological types and different host adaptations, as well as between the E. coli enterotoxin and that of Vibrio cholerae, were investigated.