Purified hepatocytes stimulate the development of L3T4-, Ly2+ allospecific cytolytic T cells from naive splenocytes after 5 days in primary mixed lymphocyte-hepatocyte culture (MLHC). Previous studies indicate that the immunogenicity of purified hepatocytes relates to the expression of MHC class I antigen. The purpose of the following experiments was to identify the cell subsets that specifically recognize hepatocyte MHC class I antigen. We employed primed lymphocyte testing (PLT) in order to test for a "second set" response. Cells from primary MLHC reverted to a functionally quiescent state when they were grown in culture for an additional 7-9 days. The cells were then tested for cytotoxicity or rechallenged with allogeneic, syngeneic, or "third party" hepatocytes and tested for proliferation. Allocytotoxicity was low on day 12 in MLHC, but the sensitized cell population demonstrated peak proliferation in response to allogeneic hepatocytes 48 hr after restimulation. When bulk PLT cells were immunodepleted, both L3T4+, Ly2- and L3T4-, Ly2+ T cell subsets demonstrated a "second set" response to allogeneic hepatocytes consistent with specific recognition of and retention of "memory" for hepatocyte MHC class I alloantigen.