It is shown that the trypsin-treatment of rat liver mitochondria, depleted of the outer membrane, causes a strong inactivation of phosphatidylserine decarboxylase. This inactivation is dependent on trypsin concentration and the time of digestion in a similar manner as the inactivation of cytochrome oxidase. Under these conditions only a moderate inactivation of succinate dehydrogenase is observed. Phosphatidylserine decarboxylase is thus localized in the outer leaflet of the inner mitochondria membrane or, at least, is accessible from the outer surface of the inner membrane.