The rhesus macaque ( Macaca mulatta) has become a popular animal model for several human infectious diseases, such as HIV (modeled by SIV infection), hepatitis, and malaria. Investigation of T-cell responses in experimental infectious diseases in rhesus macaques has benefited from an expanding understanding of the diversity of macaque MHC class I heavy chains and the restriction of antigen presentation by macaque class I molecules. Here we add to this understanding with the first nucleotide sequences of M. mulatta beta(2)-microglobulin (beta(2)m) mRNA, including a portion of the 3'-untranslated region (3'UTR). In pairwise comparison, the beta(2)m protein of M. mulatta differs from human and chimpanzee beta(2)m by nine amino-acid substitutions (92% identity), and from Macaca fascicularis by one amino-acid difference in the signal peptide region (99% identity). Allelic variations were identified at one site in the 3'UTR. A structural analysis of human or chimpanzee beta(2)m and M. mulatta beta(2)m suggests that the differences cluster in three solvent-exposed clusters and do not involve contacts with the class I heavy chain. We predict that human and macaque beta(2)m should bind interchangeably with the class I heavy chains of the other species, and show that four M. mulatta class I alleles form cell surface complexes with human beta(2)m. Further, we predict that W6/32 (a monoclonal antibody that recognizes a combined epitope of some class I heavy chains and beta(2)m with a subtle species dependence) should bind similarly human or macaque class I molecules that are bound with beta(2)m of either species, supported by evidence of recognition of both heterologous and homologous complexes of macaque class I heavy chains. Our findings contribute to the growing understanding of rhesus macaque histocompatibility antigens and antigen presentation, and to the phylogeny of beta(2)m in primates.