The relative contributions of direct and indirect pathways of allorecognition to graft rejection remain controversial. Recent reports suggest that the indirect pathway may play a prominent role in both acute and chronic allograft rejection. Such studies suggest that MHC-derived allopeptides are more immunogenic than those derived from minor histocompatibility or other nominal Ags. The aim of this study was to characterize the immunogenicity of MHC alloantigens in MHC-defined miniature swine via primary and secondary MLR culture assays. APCs were selectively depleted from either responder or stimulator cell populations to specifically analyze direct and indirect proliferative responses, respectively. Radio-resistant cytokine secretion and subsequent backstimulation of responder cells was eliminated by using stimulators that were either lysed or unresponsive to the responder MHC haplotypes. When the effect of backstimulation was eliminated from MLR culture assays, indirect proliferative responses were not observed among naive responders. Only after in vivo priming of responder animals could indirect proliferation be detected. These data do not refute the potential importance of indirect allorecognition in graft rejection. However, they suggest that MHC-derived alloantigens behave similarly in vitro to minor histocompatibility Ags, with comparable immunogenicity. These data also suggest that the MLR culture assay does not accurately reflect the importance of indirect mechanisms that have previously been reported in experimental models of graft rejection. A greater understanding of the indirect pathway and the associated immunogenicity of MHC allopeptides has the potential benefit of enabling the development of therapeutic interventions to prevent or halt allograft rejection.