The maltose transporter of Saccharomyces cerevisiae is rapidly degraded during fermentation in the absence of a nitrogen source. The location and mechanism of degradation of the transporter have been investigated. Using mutants defective in endocytosis, we have shown that degradation of this transporter requires internalization by endocytosis. In addition, studies of mutants defective in proteasome or vacuolar proteolysis revealed that degradation occurs in the vacuole and is independent of proteasome function. The results also revealed that degradation of the maltose transporter requires Sec18p and raised the question of whether in the absence of Sec18p activity the internalized maltose transporter is recycled back to the plasma membrane.Sugar transporters are integral plasma membrane proteins that catalyze the first rate-limiting step of glycolysis in Saccharomyces cerevisiae (11). Several strategies are used by this organism to adjust the activities of these proteins to different environmental conditions (22). One of these strategies is the irreversible inactivation of the transporters, which occurs when protein synthesis is impaired upon exhaustion of a nitrogen source in the medium (3-5, 9, 23, 27) and which results in dramatic physiological effects (20,21,23). This inactivation, known as catabolite inactivation (17), affects mainly the V max of the transporters, follows first-order kinetics, and is an energy-dependent process stimulated by fermentable substrates (3,4,9). By using polyclonal antibodies against a recombinant maltose transporter protein, it has been shown that catabolite inactivation is due to proteolysis (26). The experiments reported here attempt to establish the location and mechanism of the maltose transporter degradation. We investigated the inactivation of the maltose transporter by measuring the rate of maltose uptake with radioactive sugar as well as by determining the cellular content of the transporter with polyclonal antibodies. Possible loci investigated are the plasma membrane, which is the locus of transporter action, the cytoplasm, and the vacuole after internalization of the transporter by endocytosis. In this study, we used strains defective in the internalization step of endocytosis as well as strains that show a defect either in the ''chymotrypsin-like'' activity of the proteasome complex or in the two main vacuolar endopeptidases.