We have isolated a cDNA clone which encodes the Qa-1a histocompatibility Ag from a library prepared from Con A-activated B10.BR mouse spleen cells. The clone encodes a protein of 322 amino acids with three potential N-glycosylation sites. The coding sequence shows strongest similarity with that of the T23d gene of DBA/2 mice which encodes the Qa-1b molecule. Molecular modeling of the putative peptide-combining site indicates most of the differences between Qa-1a and Qa-1b are located peripheral to the binding cleft, with only two amino acid substitutions, at positions 9 and 24, which might affect peptide binding. Many features of the Qa-1 binding cleft are also conserved in the rat RTBM.1 and in human HLA-E molecules. This suggests that all of these molecules may associate with structurally similar peptides.