The magnitude of response elicited by CTL-inducing vaccines correlates with the density of MHC class I (MHC-I)-peptide complexes formed on the APC membrane. The MHC-I L chain, β 2-microglobulin (β 2 m), governs complex stability. We reasoned that genetically converting β 2 m into an integral membrane protein should exert a marked stabilizing effect on the resulting MHC-I molecules and enhance vaccine efficacy. In the present study, we show that expression of membranal human β 2 m (hβ 2 m) in mouse RMA-S cells elevates MHC-I thermal stability. RMA-S transfectants bind an exogenous peptide at concentrations 10 4-to 10 6-fold lower than parental RMA-S, as detected by complex-specific Abs and by T cell activation. Moreover, saturation of the transfectants’ MHC-I by exogenous peptide occurs within 1 min, as compared with∼ 1 h required for parental cells. At saturation, however, level of peptide bound by modified cells is only 3-to 5-fold higher. Expression of native hβ 2 m only results in marginal effect on the binding profile. Soluble β 2 m has no effect on the accelerated kinetics, but the kinetics of transfectants parallel that of parental cells in the presence of Abs to hβ 2 m. Ab inhibition and coimmunoprecipitation analyses suggest that both prolonged persistence of peptide-receptive H chain/β 2 m heterodimers and fast heterodimer formation via lateral diffusion may contribute to stabilization. In vivo, peptide-loaded transfectants are considerably superior to parental cells in suppressing tumor growth. Our findings support the role of an allosteric mechanism in determining ternary MHC-I complex stability and propose membranal β 2 m as a novel scaffold for CTL induction.