The immunogenicity of major histocompatibility complex (MHC) class I+ class II- hepatocytes is controversial. We studied the effect of pretransplant donor-specific sensitization with either purified hepatocytes (HC) or splenocytes (Spl) on subsequent skin allograft survival. Five million Percoll-purified DBA HC or 10 × 106 DBA Spl were injected into C57BL/6 recipients either intraperitoneally (ip) or into a sponge matrix allograft. Twelve days later, sensitized mice received a DBA skin graft. On the same day, allogeneic (DBA) and syngeneic (BL/6) skin grafts were placed on naive BL/6 mice. In naive BL/6 mice, allogeneic skin graft survival was 7.8 ± 0.5 days (n = 4), and syngeneic survival was indefinite (n = 5). Skin graft survival (mean ± SD in days) in recipients sensitized with hepatocytes ip was 6.0 ± 1.2 days (n = 5) compared with 5.6 ± 0.5 days in recipients sensitized with splenocytes ip. Similarly, graft survival in recipients that received hepatocytes into a sponge matrix allograft was 5.67 ± 1 days (n = 6) compared with 5.2 ± 1.1 days (n = 8) in those that received splenocytes into the sponge. There was no difference in graft survival between mice sensitized with HC vs Spl, nor between mice injected ip vs with the sponge. All sensitized mice experienced accelerated graft rejection compared with naive controls (P < 0.000). These results demonstrate that purified MHC class I+, class II- murine HCs are immunogenic in vivo. Sensitization with donor-specific HCs led to accelerated rejection of subsequent skin grafts, similar to the accelerated rejection seen after sensitization with MHC class I+ and class II+ splenocytes.