Recently, we reported that hepatic allografts are permanently accepted when transplanted after intraportal injection (IP) of donor spleen cells (SPCs). The mechanism of this effect was investigated using various protocols for antigen injection. ACI (RT1a) and Buffalo (RT1b) rats were used as donors and recipients, respectively. Experimental groups were divided into the following groups depending on treatment: IP, intravenous injection (IV), splenectomy (Spx), and intravenous injection after splenectomy (IV+Spx). Accumulation of donor antigen in the various organs was examined by injecting 51Cr-labeled SPCs. Cellular and humoral responses after SPC injection was assessed by delayed-type hypersensitivity response and complement-dependent cytotoxicity (CDC) assay. Heterotopic cardiac transplantation and orthotopic hepatic transplantation were performed 10 days after each treatment. The accumulation ratio in the liver was significantly higher in the IP and IV+Spx groups than in the IV group. Delayed-type hypersensitivity responses were lower in the IP and IV+Spx groups than in the IV or Spx groups. The CDC titer 7 days after inoculation was significantly lower in the IP and IV+Spx groups than in the IV group. In order to examine whether the treatment actively suppressed the immune response, the animals were rechallenged with SPCs given intravenously 10 days after the initial injection. Elevation of CDC titer was suppressed in the IP and IV+Spx groups, but not in the IV and Spx groups. Significant prolongation of cardiac allograft survival was not observed in the IV and Spx groups. Prolongation was observed in the IP and IV+Spx groups. The survival of hepatic allografts in the IV group was decreased. No significant prolongation of graft survival was observed in the Spx group. In contrast, all hepatic allotransplants in the IP and IV+Spx groups survived over 45 days. These findings suggest that the accumulation of donor SPCs in the liver may play an important role in inducing unresponsiveness after intraportal injection of donor SPCs.