Over 45 VPS genes (vacuolar protein sorting) in Saccharomyces cerevisiae are necessary for the correct sorting and delivery of vacuolar hydrolases. Yeast strains carrying mutations in a subset of these VPS genes (class D vps mutants) are also defective in the segregation of vacuolar material into the developing daughter cell and are morphologically characterized by having large central vacuoles. The class D VPS gene products, which include a Rab5 homologue (VPS21/YPT51) and a syntaxin homologue (PEP12/VPS6), have been proposed to function together at a particular step along the vacuolar protein sorting pathway. We have cloned another class D VPS gene, VPS45, which is homologous to a growing family of genes that encode Sec1p-like proteins. Vps45p is predicted to be a hydrophilic protein of 577 amino acids with a molecular mass of 67 kDa. Fractionation studies show that Vps45p is a peripheral membrane protein that cofractionates with Golgi-like membranes, consistent with Vps45p functioning in membrane traffic between the Golgi and the vacuole. Using a temperature-sensitive allele of VPS45, we show that inactivation of Vps45p causes the rapid accumulation of small (40-60 nm) vesicles and secretion of the vacuolar hydrolase carboxypeptidase Y. Because the entire yeast secretory pathway is functional after the temperature-induced inactivation of Vps45p, we conclude that the accumulated vesicles represent transport intermediates between the Golgi and the vacuole.