ABSTRACT In the budding yeast Saccharomyces cerevisiae , the Cdc42 effector Ste20 plays a crucial role in the regulation of filamentous growth, a response to nutrient limitation. Using the split-ubiquitin technique, we found that Ste20 forms a complex with Vma13, an important regulatory subunit of vacuolar H + -ATPase (V-ATPase). This protein-protein interaction was confirmed by a pulldown assay and coimmunoprecipitation. We also demonstrate that Ste20 associates with vacuolar membranes and that Ste20 stimulates V-ATPase activity in isolated vacuolar membranes. This activation requires Ste20 kinase activity and does not depend on increased assembly of the V 1 and V 0 sectors of the V-ATPase, which is a major regulatory mechanism. Furthermore, loss of V-ATPase activity leads to a strong increase in invasive growth, possibly because these cells fail to store and mobilize nutrients efficiently in the vacuole in the absence of the vacuolar proton gradient. In contrast to the wild type, which grows in rather small, isolated colonies on solid medium during filamentation, hyperinvasive vma mutants form much bigger aggregates in which a large number of cells are tightly clustered together. Genetic data suggest that Ste20 and the protein kinase A catalytic subunit Tpk2 are both activated in the vma13 Δ strain. We propose that during filamentous growth, Ste20 stimulates V-ATPase activity. This would sustain nutrient mobilization from vacuolar stores, which is beneficial for filamentous growth.