An efficient, single-column preparative-scale fractionation of pronase on CM-Sephadex using a linear gradient of pyridine-acetate, pH 5.0, has been developed. Under these conditions excellent resolution and minimal autolysis of the component proteolytic enzymes occurs. Only three major endopeptidases with caseinolytic activity are found. Streptomyces griseus trypsin (S.G.T.) is recovered in adequate purity for amino acid sequence studies. Streptomyces griseus Protease A and Protease B have been shown to correspond to PNPase I and II previously described by Wählby. Two aminopeptidases and a carboxypeptidase have also been demonstrated. Pronase appears to be a less complex mixture of proteolytic enzymes than had previously been appreciated.