Subunits of the mitochondrial ATP synthase complex are expressed on the surface of tumors, bind the TCR of human Vγ9/Vδ2 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 α and β chains of ecto-F1-ATPase are detected in membrane protein precipitates from immunofluorescence-negative cells, suggesting that ATPase epitopes are masked. Removal of β2-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. Upregulation of MHC-I on these cells following IFN-γ and/or TNF-α 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 β chain expression on HepG2 cells shows a colocalization 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 Vγ9/Vδ2 T cell activity through binding to natural killer inhibitory receptors.