Cyclosporin A and tacrolimus are important immunosuppressive drugs. They share a diabetogenic action as one of their most serious adverse effects. In a single study, tacrolimus (100 nM) inhibited human insulin gene transcription in the beta-cell line HIT. Using transfections of a human insulin-reporter gene into HIT cells, the present study shows that this inhibition is seen only at high concentrations of tacrolimus and is not caused by cyclosporin A. However, after stimulation by the major second messengers in the regulation of the insulin gene, cAMP and depolarization-induced calcium influx, both tacrolimus and cyclosporin A inhibited human insulin gene transcription in a concentration-dependent manner with IC(50) values of 1 nM and 30 nM, respectively. A further analysis offers a mechanism for this effect by revealing that the activation by cAMP and calcium of human insulin gene transcription is mediated by the transcription factor cAMP-responsive element binding protein (CREB) whose activity is inhibited by the immunosuppressants. These data demonstrate for the first time that cAMP- and calcium-induced activity of the human insulin gene is mediated by CREB and blocked by both tacrolimus and cyclosporin A at concentrations that inhibit calcineurin phosphatase activity. Since also the immunosuppressive effects of cyclosporin A and tacrolimus are thought to be secondary to inhibition of calcineurin, the present study suggests that inhibition of human insulin gene transcription by the immunosuppressants is clinically important and may contribute to their diabetogenic effect.