Using an immunological screening procedure that allows the detection of yeast cells aberrantly secreting vacuolar proteins, we have isolated a cloned DNA fragment containing the structural gene for the vacuolar enzyme proteinase A (PrA; EC 3.4.23.6). A large portion of PrA is misdirected to the cell surface in cells harboring the PrA structural gene on a multicopy plasmid. This mislocalized PrA traverses the late stages of the secretory pathway and differs slightly in apparent molecular weight from the vacuolar form. A deletion in the genomic copy of the PrA structural gene eliminates immunoreactive PrA as well as the enzymatic activities of at least three other vacuolar hydrolases. In the case of the vacuolar enzyme carboxypeptidase Y (EC 3.4.16.1), the lack of activity is due to the absence of proteolytic activation of the zymogen. Thus, PrA may be required for in vivo processing of a number of yeast vacuolar hydrolases.