We have previously reported that freshly isolated percoll-purified hepatocytes are MHC class I+, Class II- and stimulate allospecific cytotoxicity in mixed lymphocyte hepatocyte culture. In this report we determined the immunogenicity of purified hepatocytes in vivo using a modification of the sponge matrix allograft model. We found that hepatocytes were immunogenic in vivo because they stimulated the development of cytolytic effectors in allogeneic but not syngeneic hepatocyte sponge matrix allografts. These cytolytic effectors were not macrophages (because cytotoxicity was intact after nylon wool depletion of the bulk sponge effectors) but rather were Ly2+, L3T4- T cells. Cytolytic effectors in both MLHC and hepatocyte sponge matrix allografts demonstrated marked specificity for allogeneic MHC class I antigen of stimulator and donor hepatocytes, respectively. Allospecific cytolytic T cells developed in both MLHC and hepatocyte sponge matrix allografts across an isolated MHC class I genetic disparity but not across an isolated class II genetic disparity. Finally, sequential phenotypic analysis of host cells infiltrating allogeneic hepatocyte sponge matrix allografts demonstrated the presence of macrophages, Ly2+, L3T4- and Ly2”, L3T4+ T cells.