To aid in the identification of key residues responsible for the control of class II MHC beta-alpha dimer assembly and expression, a series of cotransfections of human plus mouse beta- and alpha-genes was performed. The resulting expression data were correlated with the sequences of the relevant proteins to identify residues that played critical roles in these processes. For the I-E/DR homologues good expression was seen for both E beta DR alpha and DR beta E alpha combinations involving several allelically variable beta-chains of each species. These results are consistent with the sequence conservation seen for I-E and DR gene products, and indicate that the species-specific differences that do exist play little role in controlling dimer formation or transport. For A beta chains, a more complex picture was seen. A beta d, but not A beta k or A beta b, was found to coexpress with human alpha-chains. Not only did A beta d show expression with the homologous DQ alpha-chain, but it also was expressed with DR alpha and DP alpha. These data indicate that species-specific residues do not control dimer expression under these conditions and confirm that allelically polymorphic residues have a crucial role in this process. Mapping studies using recombinant A beta genes established the importance of the residues in the amino-terminal half of the beta 1 domain in the differences observed among the A beta alleles. Sequence comparison of DR beta, DP beta, DQ beta, E beta, and A beta chains in this region revealed a single residue (position 12) conserved in most chains and differing in a nonconservative fashion between A beta d vs A beta b or k. A beta d has the conserved lysine at this position, whereas A beta b has methionine and A beta k has glutamine. To test whether this residue actually was important physiologically, a lysine codon was created in a recombinant A beta gene possessing the amino-terminal sequence of the kappa haplotype, and the ability of this mutant chain to be expressed with various mouse A alpha-chains was examined. This mutant chain was shown to gain the ability to be efficiently expressed with A alpha d without losing its ability to be expressed with A alpha k. These data reemphasize the special role played by allelically polymorphic residues in Ia expression and identify one such polymorphic site as position 12.(ABSTRACT TRUNCATED AT 400 WORDS)