A mutant of yeast lacking proteinase C (carboxypeptidase Y) activity has been found by using a histochemical stain to screen mutagenized colonies. This defect segregates 2:2 in meiotic tetrads. Cell extracts lacked the esterolytic, amidase, and proteolytic activities associated with proteinase C. The absence of proteinase C does not affect mitotic growth and has no obvious effect on the formation of viable ascospores or meiotic segregation. The mutant grows on peptides known to be cleaved by proteinase C in vitro. This finding is consistent with the idea that other enzymes exist in vivo with overlapping substrate specificities.