In eukaryotic cells, SNARE proteins are essential for intracellular vesicle trafficking. Several SNARE proteins are required for vacuolar protein transport and vacuolar biogenesis in Saccharomyces cerevisiae. Previously we demonstrated that one of the fission yeast SNARE proteins, Pep12p, is not required for vacuolar fusion process in Schizosaccharomyces pombe. We have re-examined the function of S. pombe Pep12p using the newly created pep12(+) deletion strain. Deletion of the fission yeast pep12(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y-and various ion- and drug-sensitivities. GFP-Pep12 fusion protein is mostly localized at the vacuolar membrane and the prevacuolar compartment. The S. pombe pep12Δ mutation phenocopies that of vps33Δ, suggesting that both Pep12p and Vps33p act at the same membrane fusion step in S. pombe, and both mutations cause vacuolar deficiency.