A rabbit antiserum "ABR2" was raised against a peptide with sequence identity to 10 amino acids of the cytoplasmic tail of HLA class I heavy chains. Western blotting and immunoprecipitation analyses demonstrate that ABR2 reacts with HLA class I heavy chains. The antiserum reacts poorly with beta 2-microglobulin (beta 2-m)-associated heavy chains and reacts strongly with free heavy chains. ABR2 reacts with immature heavy chains from the endoplasmic reticulum that have yet to bind beta 2-m and mature heavy chains that have dissociated from beta 2-m at the plasma membrane. Comparison with HC10, a mAb that recognizes an epitope defined by polymorphism at residue 62 of the alpha 1 helix of free HLA class I heavy chains, shows that ABR2 reacts with overlapping populations of free heavy chains (for those allotypes that react with both Abs), but it also identifies populations that bind to one Ab and not the other. ABR2 induces dissociation of beta 2-m from HLA-B38 molecules expressed by the human B cell line "TEM," a phenomenon not detected with other allotypes or with the same allotype in a different cell line. This study shows that association of beta 2-m with the extracellular domains of HLA class I heavy chains can cause a change in the cytoplasmic tail that prevents binding of Abs present in the ABR2 antiserum. Similar findings have been made for mouse H-2 class I molecules, which suggests that this is a general property of class I MHC molecules.