Binding of antigenic peptides to purified class I major histocompatibility complex (MHC) molecules, as measured by antigen‐specific cytolytic T lymphocyte (CTL) degranulation, was found to occur in the presence of serum but not in its absence. The role of soluble β2‐microglobulin (β2m), a normal component of serum, in class I‐peptide complex formation was therefore examined. Sera depleted of β2m did not support effective peptide binding to class I, but binding was restored in the presence of low concentrations of purified human β2m. Sequential incubation of immobilized class I with human β2m first, followed by peptide, resulted in antigenic complex formation, while reversing the order of pulsing could not. Similar results were obtained in experiments examining H‐2Db, Kb and Kd with appropriate peptides and CTL. These results demonstrate that mature class I proteins are not able to directly bind peptide, but that interaction with exogenous β2m results in a structure that will subsequently bind peptide. Binding of exogenous β2m appears to result in “empty” class I molecules, possibly by exchange for endogenous β2m, with a concomitant loss of endogenous peptide.