Using mutant strain ABYS1 of Saccharomyces cerevisiae lacking four main vacuolar proteinases, proteinase A, proteinase B, carboxypeptidase Y, and carboxypeptidase S, we examined the identities of chromatin-associated proteinases, ruling out possible contamination of the chromatin fraction by them. The chromatin of strain ABYS1 showed three peaks of proteolytic activity at pH 4, 7, and 11, and these activities were found to be derived from three species of proteinases, the aspartic, serine neutral, and serine alkaline ones. As these chromatin-associated proteinases of strain ABYS1 were identical in both quality and quantity to those of wild-type strain of yeast, we suggest that the yeast chromatin contains three species of specific proteinases as essential components.