The function of major histocompatibility complex (MHC) class I molecules is to sample peptides derived from intracellular proteins and to present these peptides to CD8+ cytotoxic T lymphocytes. In this paper, biochemical assays addressing MHC class I binding of both peptide and beta 2-microglobulin (beta 2m) have been used to examine the assembly of the trimolecular MHC class I/beta 2m/peptide complex. Recombinant human beta 2m and mouse beta 2ma have been generated to compare the binding of the two beta 2m to mouse class I. It is frequently assumed that human beta 2m binds to mouse class I heavy chain with a much higher affinity than mouse beta 2m itself. We find that human beta 2m only binds to mouse class I heavy chain with slightly (about 3-fold) higher affinity than mouse beta 2m. In addition, we compared the effect of the two beta 2m upon peptide binding to mouse class I. The ability of human beta 2m to support peptide binding correlated well with its ability to saturate mouse class I heavy chains. Surprisingly, mouse beta 2m only facilitated peptide binding when mouse beta 2m was used in excess (about 20-fold) of what was needed to saturate the class I heavy chains. The inefficiency of mouse beta 2m to support peptide binding could not be attributed to a reduced affinity of mouse beta 2m/MHC class I complexes for peptides or to a reduction in the fraction of mouse beta 2m/MHC class I molecules participating in peptide binding. We have previously shown that only a minor fraction of class I molecules are involved in peptide binding, whereas most of class I molecules are involved in beta 2m binding. We propose that mouse beta 2m interacts with the minor peptide binding (i.e. the "empty") fraction with a lower affinity than human beta 2m does, whereas mouse and human beta 2m interact with the major peptide-occupied fraction with almost similar affinities. This would explain why mouse beta 2m is less efficient than human beta 2m in generating the peptide binding moiety, and identifies the empty MHC class I heavy chain as the molecule that binds human beta 2m preferentially.