Genetically engineered structural variants of human β2-microglobulin (β2m) were produced by sequence exchange with mouse β2m for the purpose of examining species-specific antigenic determinant expression. For aggregate mapping, mouse and human β2m, which differ by 30% in their primary sequence of 99 amino acids, were prepared as chimeric (human X mouse) molecules and expressed in the FO-1 β2m-null human melanoma cell line. A chimera containing residues 1-69 from human β2m (and residues 70-99 from mouse β2m) induced expression of the epitopes defined by the anti-β2m monoclonal antibodies (mAb) BBM.1, NAMB-1, and L368; the reverse chimera did not, although HLA class I heavy chain was evident on the cell surface as determined with the TP25.99 mAb. For fine dissection of the epitopes defined by these mAbs, site-directed mutants of β2m were prepared by replacement of individual amino acids in human β2m with the dimorphic residue from mouse β2m. Substitutions were made at each divergent residue between positions 1 and 66 and, as controls for COOH-terminal modification, a series of residues between positions 75 and 94. Replacement of amino acids 38, 44, and 45, but not 16 other dimorphic residues in the linear stretch from residue 1 to residue 66, resulted in the loss of, or gross reduction in, binding by mAbs BBM.1 and NAMB-1. A reduction in binding was also observed for mAb L368. These data provide strong evidence that the antigenic epitopes defined by these mAb map to a region including S3 and its adjacent intra-β-strand turn of the three-stranded β-pleated sheet of β2m. The mapping of these epitopes is consistent with their accessibility in the assembled major histocompatibility complex class I molecule and indicates that the region from amino acid 38 to 45 is an important structural feature in the “foreignness” of human and mouse β2m.