The H+-translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of Mr approximately equal to 70,000 and approximately equal to 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-[14C]ethylmaleimide and 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole, labeled the Mr approximately equal to 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-[14C]dicyclohexylcarbodiimide labeled a polypeptide of Mr approximately equal to 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of Mr 5.2 X 10(5), 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochondrial ATPase but strongly crossreacted with antiserum against a polypeptide of Mr approximately equal to 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F0F1 ATPases.