Abstract: Yeast vacuole protein targeting (vpt) mutants exhibit defects in the sorting and processing of multiple vacuolar hydrolases. To evaluate the impact these vpt mutations have on the biogenesis and functioning of the lysosome-like vacuole, we have used light and electron microscopic techniques to analyze the vacuolar morphology in the mutants. These observations have permitted us to assign the vpt mutants to three distinct classes. The class A vpt mutants (26 complementation groups) contain 1-3 large vacuoles that are morphologically indistinguishable from those in the parental strain, suggesting that only a subset of the proteins destined for delivery to this compartment is mislocalized. One class A mutant (vpt13) is very sensitive to low pH and exhibits a defect in vacuole acidification. Consistent with a potential role for vacuolar pH in protein sorting, we found that bafilomycin A1, a specific inhibitor of the vacuolar ATPase, as well as the weak base ammonium acetate and the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, collapse the pH gradient across the vacuolar membrane and cause the missorting and secretion of two vacuolar hydrolases in wild-type cells. Mutants in the three class B vpt complementation groups exhibit a fragmented vacuole morphology. In these mutants, no large normal vacuoles are observed. Instead, many (20-40) smaller vacuole-like organelles accumulate. The class C vpt mutants, which constitute four complementation groups, exhibit extreme defects in vacuole biogenesis. The mutants lack any organelle resembling a normal vacuole but accumulate other organelles including vesicles, multilamellar membrane structures, and Golgi-related structures. Heterozygous class C zygotes reassemble normal vacuoles rapidly, indicating that some of the accumulated aberrant structures may be intermediates in vacuole formation. These class C mutants also exhibit sensitivity to osmotic stress, suggesting an osmoregulatory role for the vacuole. The vpt mutants should provide insights into the normal physiological role of the vacuole, as well as allowing identification of components required for vacuole protein sorting and/or vacuole assembly.