A method has been developed for the isolation of enterotoxin B with a purity greater than 99 and in yields of 50 to 60 from cultures of staphylococcus aureus. The method involves 1 removal of the toxin from the culture with CG-50 resin, partly neutralized at pH 6.4, and elution with 0.5 M phosphate buffer, pH 6.8 in 0.25 M sodium chloride 2 readsorption of the toxin on CG-50, partly neutralized at pH 6.8, followed by elution of the toxin with 0.15 M disodium phosphate and 3 adsorption of the toxin oncarboxymethyl cellulose and chromatographic elution with a gradient phosphate buffer 0.02 M to 0.07 M at pH 6.8. The purified toxin was eluted between 0.035 M and 0.045 M phosphate. Elution of the toxin was followed by the absorbance at 277 millimicrons and by the Oudin serological test. Final preparations were assayed in rhesus monkeys. Ultracentrifugal and electrophoretic studies show that the purified toxin is a single component with a molecular weight of approximately 35,000. Analyses show that the toxin is composed of amino acids only and therefore is a single protein.