BACKGROUND: Regulatory T cells (Tregs) are increasingly recognized as playing a major role in nondeletional tolerance. To avoid rejection before tolerance is established, clinical trials of tolerance induction include immunosuppressive drugs early posttransplant. It is therefore essential that immunosuppressive protocols do not block Tregs generation. Tregs function has been shown to depend upon interleukin-2 signaling, but there are limited data available on how calcineurin inhibitors influence Tregs development and function in vivo. METHODS: To study this, we used a previously established rat cardiac allograft model where donor-specific Tregs and tolerance are induced by pretransplant donor-specific blood transfusion (DSBT). RESULTS: In this model, we found that adjunction of 50 mg/kg cyclosporine (CsA) (not a lower dose, 10 mg/kg) at the time of DSBT (not at the time of transplantation) abrogates Tregs development and causes rejection. Interestingly, 10 mg/kg CsA given posttransplant (day 0-11) in the absence of pretransplant DSBT induced the development of Tregs and provoked a state of tolerance indistinguishable from the one induced by DSBT. Finally, DSBT given the day of transplantation did not promote tolerance, unless recipients also received a delayed short course (day 5-9) of 10 mg/kg CsA. CONCLUSIONS: Adjunction of high-dose CsA to pretransplant DSBT abrogates Tregs generation. On the contrary, a lower dose (10 mg/kg) of CsA promotes Tregs development either in synergy with perioperative DSBT (providing that a drug-free interval is respected) or by its own effect. These data provide new guidelines for a more tolerogenic use of calcineurin inhibitors in the clinic, particularly when immunomodulatory strategies aimed at inducing Tregs are applied.