2-Microglobulin (β2m) binds non-covalently to the α1, α2 and α3 domains of the α-chain of Class I major-histocompatibility-complex (MHC) molecules. On the basis of the crystal structures of human leucocyte antigens HLA-A2.1 and HLA-Aw68.1, we have used molecular-graphics analyses to define 44 contact points between 19 α-chain residues and 18 β2m residues. In 88 other α-chain sequences from the HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F and HLA-G locus products in man and the H-2, Qa and Tla loci in mouse, 37 contact sites were conserved to 90 % or more, and in β2m sequences from seven other species 40 % of contact sites were totally conserved. Four distinct regions form the contact points between the α-chain and β2m, one on each of the α1 and α2 domains and two on the α3 domain. We have further studied the α-chain sequences of three non-MHC molecules, human CDI and rat Fc receptor (FcRn), known to bind to β2m, and a third molecule, the putative product of the H301 (UL18) gene of human cytomegalovirus (CMV). CMV has been shown to bind β2m, and it has been postulated that the H301-gene product, which has sequence similarity to Class I HLA, is the protein responsible. These sequences exhibited much lower residue conservation with the MHC-linked group, although the α3 domain was the most highly conserved, and gaps and insertions were required for optimal alignments with the 90 α-chain sequences. Of the 44 β2m-α-chain contacts defined for Class I HLA, 24 α-chain contact sites were conserved in CDI, 25 in FcRn and 17 in the H301-gene product. For CDI and FcRn, the majority of the conserved β2m contacts were found in the α2 domain and the major contact region in the α3 domain. Together with the use of secondary-structure predictions, it was concluded that the binding of β2m in CDI and FcRn was MHC-like at the α3 domain, and probably also at the α2 domain for FcRn, but non-MHC-like for the α1 domain of both molecules and the α2 domain of CDI. In the H301-gene product sequence, only the β2m contacts with the main region of the α3 domain were noticeably conserved. Structural evidence for β2m binding in H301 in this α3 contact region was, however, much weakened by a sequence insertion, the presence of proline residues, and marked deviations in the secondary-structure predictions. If the H301-gene product is responsible for the binding of β2m to cytomegalovirus, this occurs in a non-MHC-like manner.