Lymphoid cells from beta 2-microglobulin (beta 2m) knockout mice transgenic for human (h) beta 2m (C57BL/10 m beta 2m-/h beta 2m+) were compared with normal mice for their binding to exogenously added h beta 2m, binding to a H-2Db peptide and for functional activity in a one-way allogenic MLC. Based on data from cellular binding studies, Scatchard analyses and flow cytometry, it is concluded that exogenous h beta 2m does not bind to hybrid MHC class I (MHC-I) molecules composed of mouse heavy chain/h beta 2m molecules expressed on lymphocytes of transgenic mice. Immunoprecipitation and SDS-PAGE analysis of metabolically labelled normal C57BL/6 lymph node cells showed binding of exogenous h beta 2m to MHC-I, in particular, to the H-2Db molecule through an exchange with endogenous mouse beta 2m. In contrast to normal H-2Db molecules, hybrid H-2Db expressed on the surface of transgenic lymphocytes binds radiolabelled peptide in the absence of exogenous added h beta 2m suggesting that a stable fraction of hybrid H-2Db molecules is empty or contain peptides with very low affinity. In a one-way allogenic mixed lymphocyte culture, transgenic splenocytes were found to be far less stimulatory than normal splenocytes. In contrast, transgenic alloreactive cytotoxic T lymphocytes developed earlier in MLC than their non-transgenic counterparts. These data indicate that the hybrid mouse heavy chain/h beta 2m complex alters the alloantigenic repertoire and influences important aspects of T-cell activation.