The filamentous fungus Neurospora crassa has many small vacuoles which, like mammalian lysosomes, contain hydrolytic enzymes. They also store large amounts of phosphate and basic amino acids. To generate an acidic interior and to drive the transport of small molecules, the vacuolar membranes are densely studded with a proton-pumping ATPase. The vacuolar ATPase is a large enzyme, composed of 8-10 subunits. These subunits are arranged into two sectors, a complex of peripheral subunits called V1 and an integral membrane complex called V0. Genes encoding three of the subunits have been isolated. vma-1 and vma-2 encode polypeptides homologous to the alpha and beta subunits of F-type ATPases. These subunits appear to contain the sites of ATP binding and hydrolysis. vma-3 encodes a highly hydrophobic polypeptide homologous to the proteolipid subunit of vacuolar ATPases from other organisms. This subunit may form part of the proton-containing pathway through the membrane. We have examined the structures of the genes and attempted to inactivate them.