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Rabbit anti-thymocyte globulins (rATG) induce CD4+CD25+forkhead box P3 (FoxP3+) regulatory T cells that control alloreactivity. In the present study, we investigated whether rATG convert T cells into functional CD4+CD25+FoxP3+CD127−/low regulatory T cells in the presence of drugs that may hamper their induction and function, i.e. calcineurin inhibitors. CD25neg T cells were stimulated with rATG or control rabbit immunoglobulin G (rIgG) in the absence and presence of tacrolimus for 24 h. Flow cytometry was performed for CD4, CD25, FoxP3 and CD127 and the function of CD25+ T cells was examined in suppression assays. MRNA expression profiles were composed to study the underlying mechanisms. After stimulation, the percentage CD4+CD25+FoxP3+CD127−/low increased (from 2% to 30%, mean, P < 0·01) and was higher in the rATG samples than in control rIgG samples (2%, P < 0·01). Interestingly, FoxP3+T cells were also induced when tacrolimus was present in the rATG cultures. Blockade of the interleukin (IL)-2 pathway did not affect the frequency of rATG-induced FoxP3+ T cells. The rATG tacrolimus-induced CD25+ T cells inhibited proliferative responses of alloantigen-stimulated effector T cells as vigorously as rATG-induced and natural CD4+CD25+FoxP3+CD127−/low T cells (67% ± 18% versus 69% ± 16% versus 45% ± 20%, mean ± standard error of the mean, respectively). At the mRNA-expression level, rATG-induced CD25+ T cells abundantly expressed IL-10, IL-27, interferon (IFN)-γ, perforin and granzyme B in contrast to natural CD25+ T cells (all P = 0·03), while FoxP3 was expressed at a lower level (P = 0·03). These mRNA data were confirmed in regulatory T cells from kidney transplant patients. Our findings demonstrate that tacrolimus does not negatively affect the induction, phenotype and function of CD4+CD25+ T cells, suggesting that rATG may induce regulatory T cells in patients who receive tacrolimus maintenance therapy.