We have previously demonstrated that the Pichia pastoris Vac8-GFP fusion protein localizes to the vacuole by in situ fluorescence microscopy, and is required for the homotypic fusion of the sequestering arms of the vacuole during microautophagy. In this study we have employed subcellular fractionation to show that PpVac8-GFP and several mutant forms of the protein co-fractionate with the P13 vacuolar membrane fraction to corroborate the previous microscopy results. This membrane association was probed by differential extraction followed by denaturing gel electrophoresis and Western blot analysis. PpVac8-GFP remained associated with the vacuolar membrane under all extraction conditions employed, including treatment of the P13 fraction with high concentrations of NaCl, urea, or detergents. We are currently attempting further analysis of the vacuolar membrane association using an enriched vacuole membrane preparation obtained by centrifugation through a Ficoll density gradient and exploring the nature of the detergent resistant association of PpVac8-GFP.