N-Ethylmaleimide (NEM) inhibited the H(+)-ATPase (EC 3.6.1.35) from Kluyveromyces lactis with a second-rate constant of 200 M-1 min-1. H(+)-ATPase was partially protected by Mg-ADP. Low concentrations of Mg protected ATPase from the effects of NEM, while high Mg sensitized ATPase to NEM. The reaction of 14C-NEM with the native enzyme modified three cysteine residues/monomer, two of which were involved in 80% of the inactivation of the enzyme. In the presence of Mg-ADP, NEM binding to the first residue had only a slight effect on the activity (10-20% inhibition). After further incubation, the modification of a second cysteine residue (probably cys-221) inactivated the ATPase. Methyl methanethiosulfonate did not inhibit the H(+)-ATPase but resulted in a NEM-resistant H(+)-ATPase. There seems to be at least one cys (probably cys-532) at, or near, the nucleotide binding site of the H(+)-ATPase, which does not appear to be essential for activity. Modification of a second cys residue (cys-221) also resulted in inactivation by NEM; this residue was not protected by ADP and thus probably is not at the ATP binding site.