Beta-2 microglobulin (beta2m)has been shown to have an effect on the structural and functional constraints that facilitate proper class I antigen presentation. To date, no evidence has pinpointed the beta2m-specific amino acids that play an integral role in affecting structure in and around the peptide binding region of class I. To delineate beta2m amino acid positions that affect the alpha-1 helical region, we generated a series of mutant beta2m proteins bearing precise amino acid substitutions. The amino acid positions chosen were based upon previous results which demonstrated that human beta2m association with H2-Ld altered the structure of the alpha-1/alpha-2 super-domain. beta2m mutant proteins were used in beta2m exchange assays with cells expressing H2-Ld. Following exchange, cells were assayed to determine whether mutant beta2m association resulted in structural alteration of class I extracellular domains. The alteration in H2-Ld structure was evidenced by an increase in the binding of an antibody (34-1-2), specific for the alpha-1 helical region of H2-Ld. Results demonstrated that amino acid substitutions in beta2m positions 33 and 53 led to a dramatic increase in the reactivity of the alpha-1 domain-specific antibody 34-1-2. Identifying beta2m amino acid positions that influence the structure of the peptide binding region may allow for a better understanding of cellular immune responses that center upon class I/beta2m expression.