Currently, aldosterone is believed to be involved in the development of cardiovascular injury as a potential cardiovascular risk hormone. However, its exact cellular mechanism(s) remains obscure. This study was undertaken to examine the effect of aldosterone on superoxide production in cultured rat aortic endothelial cells (RAEC) with possible involvement of the small GTP-binding (G) protein Rac1. The aldosterone levels showed a time-dependant (6–24 h) and dose-dependant (10^–8–10^–6 M) increase in superoxide generation, whose effect was abolished by mineralocorticoid receptor (MR) antagonist (eplerenone), Src inhibitor (PP2) and NAD(P)H oxidase inhibitor (apocynin). Aldosterone activated NADP(H) oxidase and Rac1, whose effects were abolished by eplerenone. The aldosterone-induced superoxide generation was abolished either by non-selective small G protein inhibitor (Clostridium difficile toxin A) or dominant-negative Rac1. Dominant negative Rac1 also inhibited aldosterone-induced ACE gene expression. Thus, the present study is the first to demonstrate that aldosterone induces superoxide generation via MR-mediated activation of NAD(P)H-oxidase and Rac1 in endothelial cells, thereby contributing to the development of aldosterone-induced vascular injury.