We have examined the structure of the vacuolar ATPase of Neurospora crassa using negatively stained preparations of vacuolar membranes and of detergent-solubilized and gradient-purified ATPase complexes. We also examined the peripheral sector (V1) of the enzyme after it had been removed and purified. Using different stains, vacuolar membranes displayed ball-and-stalk structures similar to those of the intact mitochondrial ATPase. However, the vacuolar ATPase was clearly different from the mitochondrial ATPase in both size and structural features. The vacuolar enzyme had a much larger head domain with a distinct cleft down the middle of the complex. This domain was held above the membrane by a prominent stalk. Most intriguing was the presence of basal components. These structures appeared to project from the vacuolar membrane near the base of the stalks. Detergent-solubilized, gradient-purified ATPases displayed the same head, stalk, and basal features as those found with the intact enzyme on vacuolar membranes. The mitochondrial ATPase was significantly smaller, and no clefted head domains or basal components were observed. When V1 and F1 particles were directly compared, a significant difference in size and shape between these two soluble ATPase sectors was apparent. V1 retained all of the features seen in the globular head of the intact complex: V-shaped, triangular, and square forms around a stain-filled core.