Abstract: Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall, we refined techniques for the purification of 'proteomics-grade' intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathione-bimane throughout the fractionation procedure, the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extravacuolar proteinase K, and the results of flow cytometric, western and marker enzyme activity analyses, vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these, 260 were identified by peptide mass fingerprinting and peptide sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified in this way, many of which correspond to alternate size and charge states of the same parent translation product, can be assigned to 117 unique ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacuolar proteins of unknown function, or other proteases, glycosidases, lipid-binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar preparations, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and/or salvage purposes.