HLA-F has low levels of polymorphism in humans and is highly conserved among primates, suggesting a conserved function in the immune response. In this study, we probed the structure of HLA-F on the surface of B lymphoblastoid cell lines and activated lymphocytes by direct measurement of peptide binding to native HLA-F. Our findings suggested that HLA-F is expressed independently of bound peptide, at least in regard to peptide complexity profiles similar to those of either HLA-E or classical MHC class I (MHC-I). As a further probe of native HLA-F structure, we used a number of complementary approaches to explore the interactions of HLA-F with other molecules, at the cell surface, intracellularly, and in direct physical biochemical measurements. This analysis demonstrated that HLA-F surface expression was coincident with MHC-I H chain (HC) expression and was downregulated upon perturbation of MHC-I HC structure. It was further possible to directly demonstrate that MHC-I would interact with HLA-F only when in the form of an open conformer free of peptide and not as a trimeric complex. This interaction was directly observed by coimmunoprecipitation and by surface plasmon resonance and indirectly on the surface of cells through coincident tetramer and MHC-I HC colocalization. These data suggest that HLA-F is expressed independently of peptide and that a physical interaction specific to MHC-I HC plays a role in the function of MHC-I HC expression in activated lymphocytes.