HLA-F is currently the most enigmatic of the human MHC-encoded class Ib genes. We have investigated the expression of HLA-F using a specific Ab raised against a synthetic peptide corresponding to amino acids 61-84 in the alpha1 domain of the predicted HLA-F protein. HLA-F is expressed as a beta2-microglobulin-associated, 42-kDa protein that shows a restricted tissue distribution. To date, we have detected this product only in peripheral blood B cells, B cell lines, and tissues containing B cells, in particular adult tonsil and fetal liver, a major site of B cell development. Thermostability assays suggest that HLA-F is expressed as an empty heterodimer devoid of peptide. Consistent with this, studies using endoglycosidase-H and cell surface immunoprecipitations also indicate that the overwhelming majority of HLA-F contains an immature oligosaccharide component and is expressed inside the cell. We have found that IFN-gamma treatment induces expression of HLA-F mRNA and HLA-F protein, but that this does not result in concomitant cell surface expression. HLA-F associates with at least two components of the conventional class I assembly pathway, calreticulin and TAP. The unusual characteristics of the predicted peptide-binding groove together with the predominantly intracellular localization raise the possibility that HLA-F may be capable of binding only a restricted set of peptides.