Posttransplantation diabetes mellitus (PTDM) is a frequent complication in immunosuppressive therapy. To better understand the molecular events associated with PTDM we investigated the effect of cyclosporine on expression and activity of hepatic nuclear factor (HNF)1alpha and 4alpha and on genes coding for glucose metabolism in cultures of the rat insulinoma cell line INS-1E, the human epithelial cell line Caco-2 and with Zucker diabetic fatty (ZDF) rats. In the pancreas of untreated but diabetic animals expression of HNF4alpha, insulin1, insulin2 and of phosphoenolpyruvate carboxykinase was significantly repressed. Furthermore, cyclosporine treatment of the insulinoma-1E cell line resulted in remarkable reduction in HNF4alpha protein and INS1 as well as INS2 gene expression, while transcript expression of HNF4alpha, apolipoprotein C2, glycerolkinase, pyruvatekinase and aldolase B was repressed in treated Caco-2 cells. Furthermore, with nuclear extracts of cyclosporine treated cell lines protein expression and DNA binding activity of hepatic nuclear factors was significantly repressed. As cyclosporine inhibits the calcineurin dependent dephosphorylation of nuclear factor of activated T-cells (NFAT) we also searched for binding sites for NFAT in the pancreas specific P2 promoter of HNF4alpha. Notably, we observed repressed NFAT binding to a novel DNA binding site in the P2 promoter of HNF4alpha. Thus, cyclosporine caused inhibition of DNA binding of two important regulators for insulin signaling, i.e. NFAT and HNF4alpha. We further investigated HNF4alpha transcript expression and observed >200-fold differences in abundance in n = 14 patients. Such variability in expression might help to identify individuals at risk for developing PTDM. We propose cyclosporine to repress HNF4alpha gene and protein expression, DNA-binding to targeted promoters and subsequent regulation of genes coding for glucose metabolism and of pancreatic beta-cell function.