Site-specific antibodies to HLA class I molecules have been raised in rabbits immunized with a synthetic peptide with the same amino acid sequence as HLA residues 328-338, which corresponds to the highly conserved intracytoplasmic region. Antibodies were detected by radioimmunoassay and were able to recognize isolated HLA heavy chains blotted onto nitrocellulose as well as the biosynthetically labeled HLA-beta 2 microglobulin complexes solubilized by non-ionic detergents. The intracellular localization of the determinants recognized by the antibodies was shown by indirect immunofluorescence labeling and the specificity of the reaction confirmed by its inhibition with the synthetic peptide. No cross-reaction was seen with H-2 antigens on murine cells. These antibodies will be important for further characterization of HLA antigens and detection of their expression in mouse cells transfected with human genes.