Mouse beta 2-microglobulin (beta 2m) is polymorphic. Sequences of five allelic wild mouse B2m genes have been determined from the large exons of genomic DNA using the polymerase chain reaction. Relative to the standard B2m(a) allele, the products of four alleles of Mus musculus origin (w2, w3, w4, and w5), differ by only one or two amino acids. w5 has a single nucleotide change, Asp85-->Val, and is identical to the c allele. w3 has two changes, Val(-13)-->Ile and Lys44-->Glu. w2 differs at Arg81-->Thr and w4 at His34-->Gln, and they share the Asp85-->Val change with B2mc and B2mw5. w5 and c cells are lysed by S19.8, a monoclonal antibody specific for beta 2mb (Ala85), in a complement-mediated cytotoxicity assay, whereas w4 cells are not. Thus, distant changes appear to introduce subtle conformational effects on beta 2m structure. Five independent isolates of Mus spretus (w1) differ the most from B2m(a), with 12 amino acid changes and only one silent substitution. Replacements predicted from the nucleotide sequence occur in loops of the molecule facing away from the class I heavy chain and not in regions where beta 2m associates with class I alpha 3 domains. Concordantly, the w1-5 allelic forms of beta 2m associate well with H-2 heavy chains. The many amino acid changes in the spretus sequence and the paucity of silent substitutions suggest that B2m has been subject to positive selection.