Although a short course of methotrexate (MTX) has a potential immunoregulatory effect on clinical allograft rejection, little data are available about the drug, and the mechanism of hyporeactivity after withdrawal is still unknown. In previous studies, we achieved permanent graft acceptance through administration of a short course of high-dose MTX during heterotopic ratheart transplantation (HHT) in a combination of DA (MHC haplotype; RT1(a)) to PVG/c (RT1(c)) rats. A 3-week course of MTX (0.25 mg/kg/day) was administered intraperitoneally to the PVG/c recipients of a DA heart graft, and 11 of 16 rats survived longer than 300 days after HHT. The splenic lymphocytes obtained from one recipient showed high reactivity against donor type splenic lymphocytes, but others did not. All serum samples from recipients showed immunosuppressive activity. The serum had anti-donor antibodies. These results showed that tolerance induced by short-course MTX was maintained by a serologic factor believed to be anti-idiotypic antibodies.