Subunits of the mitochondrial ATP synthase complex are expressed on the surface of tumors, bind the TCR of human Vgamma9/Vdelta2 lymphocytes and promote their cytotoxicity. Present experiments show that detection of the complex (called ecto-F1-ATPase) at the cell surface by immunofluorescence correlates with low MHC-class I antigen expression. Strikingly, the alpha and beta chains of ecto-F1-ATPase are detected in membrane protein precipitates from immunofluorescence-negative cells, suggesting that ATPase epitopes are masked. Removal of beta2-microglobulin by mild acid treatment so that most surface MHC-I molecules become free heavy chains reveals F1-ATPase epitopes on MHC-I+ cell lines. Ecto-F1-ATPase is detected by immunofluorescence on primary fibroblasts which express moderate levels of MHC-I antigens. Up-regulation of MHC-I on these cells following IFN-gamma and/or TNF-alpha treatment induces a dose-dependent disappearance of F1-ATPase epitopes. Finally, biotinylated F1-ATPase cell surface components co-immunoprecipitate with MHC-I molecules confirming the association of both complexes on Raji cells. Confocal microscopy analysis of MHC-I and ecto-F1-ATPase beta chain expression on HepG2 cells shows a co-localization of both complexes in punctate membrane domains. This demonstrates that the TCR target F1-ATPase is in close contact with MHC-I antigens which are known to control Vgamma9/Vdelta2 T cell activity through binding to natural killer inhibitory receptors.