Studies suggest that immunosuppression associated with liver transplantation may be related to the secretion of MHC class I antigen (Ag) by hepatocytes. To investigate this possibility, we developed a culture system whereby naive Lewis (RT1.A1) splenocytes were cocultured with autologous hepatocytes transfected with plasmids encoding either the membrane-bound or secreted allogeneic MHC class I Ag, RT1.Aa. Cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) limiting dilution assays were subsequently performed on preconditioned lymphocytes. Lymphocytes preconditioned with hepatocytes secreting RT1.Aa showed an alloantigen specific inhibition of CTL precursors (CTLp). In contrast, exposure of splenocytes to hepatocyte-expressed membrane-bound RT1.Aa resulted in Ag-specific CTLp priming. This CTLp priming effect by hepatocyte-expressed membrane-bound Ag could be effectively blocked when splenocytes were first preincubated with hepatocytes secreting RT1.Aa, before being exposed to hepatocytes expressing membrane-bound RT1.Aa. In contrast to CTLp, HTLp frequency, as determined by IL-2 production, was unaffected by either hepatocyte-expressed membrane-bound or secreted RT1.Aa. Further studies on splenocytes conditioned with hepatocytes expressing secreted allo-MHC Ag suggest the possibility of suppressor cell development. This was demonstrated by prolongation of ACI (RT1a) heart allograft survival in Lewis recipients following adoptive transfer of splenocytes that were preconditioned in vitro with hepatocytes secreting alloantigen.