1. Cyclosporine A (CsA) increases eNOS mRNA expression in bovine cultured aortic endothelial cells (BAEC). As some effects of CsA may be mediated by reactive oxygen species (ROS), present experiments were devoted to test the hypothesis that the CsA-induced eNOS up-regulation could be dependent on an increased synthesis of ROS. 2. CsA induced a dose-dependent increase of ROS synthesis, with the two fluorescent probes used, DHR123 (CsA 1 microM: 305+/-7% over control) and H2DCFDA (CsA 1 microM: 178+/-6% over control). 3. Two ROS generating systems, xanthine plus xanthine oxidase (XXO) and glucose oxidase (GO), increased the expression of eNOS mRNA in BAEC, an effect which was maximal after 8 h of incubation (XXO: 168+/-21% of control values. GO: 208+/-18% of control values). The ROS-dependent increased eNOS mRNA expression was followed by an increase in eNOS activity. 4. The effect of CsA on eNOS mRNA expression was abrogated by catalase, and superoxide dismutase (SOD). In contrast, the antioxidant PDTC augmented eNOS mRNA expression, both in basal conditions and in the presence of CsA. 5. The potential participation of the transcription factor AP-1 was explored. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. 6. The present results support a role for ROS, particularly superoxide anion and hydrogen peroxide, as mediators of the CsA-induced eNOS mRNA up-regulation. Furthermore, they situate ROS as potential regulators of gene expression in endothelial cells, both in physiological and pathophysiological situations.