BACKGROUND: We have shown previously that a 2-week course of leflunomide (LF) together with a maintenance therapy of cyclosporine (CsA) rendered hamster-to-rat heart xenografts (Xg) resistant against anti-hamster IgM xenoantibody (XAb)-mediated rejection, a state compatible with the notion of accommodation. Our aim in this study was to investigate the mechanism underlying this Xg accommodation. METHODS: "Accommodated" Xgs were retransplanted to CsA-treated naive rats in the presence or absence of additional LF treatment or anti-hamster IgM serum injection. Immunohistopathology and fluorescence-activated cell sorting was performed to detect IgM and complement (C) deposition in Xgs, and endothelial cell (EC) expression of P- and E-selectin, ICAM-1, and VCAM-1 in vivo and in vitro. RESULTS: Retransplanted accommodated Xgs were rejected in CsA-treated naive rats and elicited IgM XAbs. Passive transfer of IgM XAbs provoked hyperacute rejection of both control and retransplanted Xgs. Addition of a 5-day course of LF prevented the rejection of only accommodated Xgs. Adoptively transferred IgM XAbs were deposited in rejected control and accommodated Xgs, but not in accommodated Xgs accepted by LF-treated rats. LF blocked the EC induction of P- and E-selectins in both control fresh and accommodated Xgs. Hence, after retransplantation accommodated Xgs express mainly induced xenoantigens (XAgs), such as P- and E-selectins, that can entirely be suppressed by LF. In contrast, control hamster Xgs express additional XAgs and remain susceptible to XAb-mediated rejection. These findings are in agreement with in vitro studies showing that LF totally suppressed induced EC antigens (e.g., P-selectin and E-selectin), but not constitutively expressed antigens (e.g., ICAM-1). CONCLUSION: Accommodated Xgs show a down-regulation of constitutive XAgs, but may be rejected after retransplantation by a mechanism involving EC expression of inducible XAgs. LF is able to block this latter XAg induction.