The monoclonal antibody W6/32 is one of the most commonly used pan‐HLA‐ABC antibodies in studying human MHC I structure and function. We have discovered that the reactivity of W6/32 is absolutely sensitive to the amino terminus of human β₂‐microglobulin (hβ₂m). Bac‐terially expressed recombinant forms of hβ₂m that have been extensively used in structural and biochemical studies of MHC I molecules often have an additional methionine at their amino terminus. Cell surface MHC I molecules reconstituted with allele‐specific peptides and recombinant hβ₂m are reactive with various HLA‐specific mAbs, but not W6/32. In contrast, cell surface HLA molecules reconstituted with peptide and native hβ₂m, which has no amino terminal methionine, are recognized by W6/32 as well as other HLA‐specific mAbs. Thus, the specificity of W6/32 includes the amino terminus of hβ₂m.