While studying the involvement of cyclic adenosine 3',5'-monophosphate (cAMP) in the fluid secretion caused by heat-stable enterotoxin (ST) from Escherichia coli P16 in infant mice, it was noted that the culture filtrate containing ST also contained large amounts of cAMP. The present paper details attempts to obtain a cAMP-free ST preparation. The organisms were grown in a defined medium, and the heated culture filtrate was concentrated by reverse osmosis. After methanol extraction of the filtrate, which removed 80% of the nonactive solids, the methanol-soluble ST was further purified by gel filtration through a Sephadex G-10 column. The first fraction recovered after gel chromatography contained ST with a negligible amount of cAMP. Treatment with methanol did not adversely affect the enterotoxic activity. Certain parameters of the infant mouse model have been investigated, and using our ST preparation it has been found that animals remain responsive up to 15 days of age with an optimum assay time of 2 h after toxin challenge.