The low-molecular-weight, heat-stable type of Escherichia coli enterotoxin (ST) was obtained from sterile syncase broth filtrates of human and animal enteropathogenic E. coli strains. ST was assayed in infant mice, and it was found that this assay was specific for ST in that the high-molecular-weight, heat-labile type of E. coli exterotoxin would not cause fluid accumulation in these animals. ST was first detected in the broth of a culture of human strain 334A(015:H11) 5 h after inoculation, immediately after the log phase of growth; the highest concentration of ST occurred at 24 h after inoculation. ST was purified 13-fold by ultrafiltration and gel filtration chromatography. The purest preparation contained 15% protein and 2% carbohydrate. The mean effective dose or 1 unit dose in infant mice was 5.0 mug/mouse. The ST molecular weight was between 1,000 and 10,000, and its activity was resistant to acid, trypsin, and Pronase.