BACKGROUND: It was recently shown that leflunomide (LF) delayed xenoantibody (XAb) formation and xenograft (Xg) rejection in a hamster-to-rat heart transplantation model. Our aim in this study was further investigation of the mechanism of LF-mediated suppression of XAb formation. METHODS: Hamster hearts were heterotopically transplanted to euthymic or nude rats receiving LF and/or cyclosporine (CsA). Second hamster hearts were transplanted at the time of first Xg rejection. Serum from rejecting rats was transferred to naive rats receiving a hamster heart Xg. The isotype of XAbs was examined by fluorescence-activated cell sorting. Tissue deposition of XAbs and complement was determined by immunofluorescence. XAb formation and its response to LF were also investigated in severe combined immunodeficient mice reconstituted with purified CD5+ or CD5- rat B cells. RESULTS: After xenografting, untreated PVG rats developed high titers of anti-hamster IgM XAbs that appeared T-independent (T-I) as they could not be suppressed by CsA and also occurred in athymic nude rats. A second Xg transplanted in control or CsA-treated rats rejecting a first Xg was subject to hyperacute rejection. Hyperacute rejection also occurred in naive rats after adoptive transfer of serum from rejecting rats. Monotherapy with LF resulted in a suppression of early IgM XAb formation and in a delay of Xg rejection, which was associated with predominantly IgG anti-hamster XAbs. These XAbs were T-dependent, as they did not occur in nude rats and were suppressed by CsA. CD5+ B lymphocytes appeared to contribute to T-I IgM XAb formation, as LF reduced the percentage of peripheral blood CD5+ B lymphocytes and severe combined immunodeficient mice reconstituted with purified CD5+ B cells, but not with CD5- B cells, produced anti-hamster IgM which were suppressed by LF but not CsA. CONCLUSIONS: In rats, T-I XAb formation is a first step leading to hamster Xg rejection and is suppressed by LF leading to prolonged Xg survival.