The plasma membrane H(+)-ATPase of Neurospora crassa was treated with 5,5'-dithiobis(2-nitrobenzoate) to determine its cysteine content and with 2-nitro-5-thiosulfobenzoate to determine its cystine content. Six and seven mol of thiols/mol of H(+)-ATPase were detected in the 5,5'-dithiobis(2-nitrobenzoate) and 2-nitro-5-thiosulfobenzoate reactions, respectively, indicating that 6 of the 8 cysteine residues in the molecule are present as free cysteines and that 2 are present in disulfide linkage. The results of quantitative carboxymethylation experiments using [14C]iodoacetate under nonreducing and reducing conditions fully support this conclusion. Preparations of the ATPase 14C carboxymethylated under the above conditions were treated with trypsin, and the tryptic digests were resolved into hydrophilic and hydrophobic peptide fractions by our recently published procedure (Rao, U.S., Hennessey, J.P., Jr., and Scarborough, G.A. (1988) Anal. Biochem. 173, 251-264). Five of the six labeled free cysteine peptides partitioned into the hydrophilic peptide fraction and were purified and established to contain Cys376, Cys409, Cys472, Cys532, and Cys545. The labeled free cysteine residue in the hydrophobic peptide fraction was identified as either Cys840 or Cys869 by virtue of its presence in a large approximately 21-kDa hydrophobic peptide established previously to begin at Ser660. This in turn identified either Cys840 or Cys869 as one of the disulfide bridge cysteines. The other disulfide bridge cysteine was identified as Cys148 by purification and NH2-terminal sequencing of an additional peptide labeled in the reduced enzyme. The disulfide bridge is therefore between Cys148 and either Cys840 or Cys869. Because Cys148 is present in a putative membrane-embedded sector near the NH2 terminus of the ATPase molecule and Cys840 and Cys869 are present in a similar sector near the COOH terminus, it is possible that the disulfide bridge plays an important structural role in holding the two major membrane-embedded sectors of the molecule, distant in the linear sequence, together.