Disruption of vacuolar biogenesis in the pathogenic yeast Candida albicans causes profound defects in polarized hyphal growth. However, the precise vacuolar pathways involved in yeast-hypha differentiation have not been determined. Previously we focused on Vps21p, a Rab GTPase involved in directing vacuolar trafficking through the late endosomal prevacuolar compartment (PVC). Herein, we identify two additional Vps21p-related GTPases, Ypt52p and Ypt53p, that colocalize with Vps21p and can suppress the hyphal defects of the vps21⌬/⌬ mutant. Phenotypic analysis of gene deletion strains revealed that loss of both VPS21 and YPT52 causes synthetic defects in endocytic trafficking to the vacuole, as well as delivery of the virulence-associated vacuolar membrane protein Mlt1p from the Golgi compartment. Transcription of all three GTPase-encoding genes is increased under hyphal growth conditions, and overexpression of the transcription factor Ume6p is sufficient to increase the transcription of these genes. While only the vps21⌬/⌬ single mutant has hyphal growth defects, these were greatly exacerbated in a vps21⌬/⌬ ypt52⌬/⌬ double mutant. On the basis of relative expression levels and phenotypic analysis of gene deletion strains, Vps21p is the most important of the three GTPases, followed by Ypt52p, while Ypt53p has an only marginal impact on C. albicans physiology. Finally, disruption of a nonendosomal AP-3-dependent vacuolar trafficking pathway in the vps21⌬/⌬ ypt52⌬/⌬ mutant, further exacerbated the stress and hyphal growth defects. These findings underscore the importance of membrane trafficking through the PVC in sustaining the invasive hyphal growth form of C. albicans. The fungal vacuole plays a vital role in supporting the pathogenicity of Candida albicans and Cryptococcus neoformans (1-3) and is likely to be critical for other fungal pathogens to cause disease (4, 5). In both C. albicans and C. neoformans, defects in vacuolar function result in a number of pathogenesis-related phenotypes, including susceptibility to host-related stresses and loss of virulence-related attributes (1, 2, 6-8). Vacuole-deficient C. albicans mutants have profound defects in invasive hyphal growth (3, 6, 9), which is crucial for virulence (10, 11).Gow and Gooday first observed that the fungal vacuole expands dramatically during C. albicans hyphal growth to yield extensively vacuolated subapical compartments (12). While several subsequent studies have established that vacuolar biogenesis plays a crucial role in supporting polarized hyphal growth, the contribution of individual trafficking pathways is unknown. Multiple vacuolar trafficking pathways have been identified in the model yeast Saccharomyces cerevisiae (13). Several vacuolar proteins, including carboxypeptidase Y, are delivered to the vacuole through a major biosynthetic trafficking route from the Golgi apparatus via the late endosome (also known as the prevacuole compartment [PVC]). This endosomal route is dependent upon the Rab GTPase Vps21p (14-16). A second Golgi-to-vacuol...