When pig stomach membrane H+,K(+)-ATPase preparations were incubated with [gamma-32P]ATP and Mg2+ with vanadate, 32P was incorporated into the alpha-chain of H+,K(+)-ATPase to a steady-state level of approximately 0.7 mol of phosphotyrosine (Tyr(P))/mol of phosphoenzyme intermediates. The addition of a membrane H+,K(+)-ATPase preparation with Mg2+ accelerated the liberation of 32P from Tyr(P) residues in the alpha-chain. Mild tosylphenylalanyl chloromethyl ketone-trypsin treatment solubilized 32P-containing peptides from the alpha-chain almost completely. A reverse-phase column chromatography of the supernatant gave two peaks of 32P-peptide with similar total radioactivities. The amino acid sequence of both peaks was shown to be Gly-Lys-Ala-Glu-Asn-Tyr-Glu-Leu-Tyr-Gln--, which is consistent with the amino-terminal sequence of the alpha-chain of H+,K(+)-ATPase deduced from cDNA from pig stomach except that the initial Met was absent. The comparison of the recovery of amino acid from each Edman cycle showed that the phosphorylation of Tyr10 occurred preceding the phosphorylation of Tyr7. These data and others suggested the presence of a novel membrane-bound enzyme system to participate in reversible phosphorylation of both Tyr residues in the alpha-chain of H+,K(+)-ATPase.