In Saccharomyces cerevisiae the vacuolar protein aminopeptidase I (API) is localized to the vacuole independent of the secretory pathway. The alternate targeting mechanism used by this protein has not been characterized. API is synthesized as a 61-kD soluble cytosolic precursor. Upon delivery to the vacuole, the amino-terminal propeptide is removed by proteinase B (PrB) to yield the mature 50-kD hydrolase. We exploited this delivery-dependent maturation event in a mutant screen to identify genes whose products are involved in API targeting. Using antiserum to the API propeptide, we isolated mutants that accumulate precursor API. These mutants, designated cvt, fall into eight complementation groups, five of which define novel genes. These five complementation groups exhibit a specific defect in maturation of API, but do not have a significant effect on vacuolar protein targeting through the secretory pathway. Localization studies show that precursor API accumulates outside of the vacuole in all five groups, indicating that they are blocked in API targeting and/or translocation. Future analysis of these gene products will provide information about the subcellular components involved in this alternate mechanism of vacuolar protein localization.