Aldosterone and NaCl can have adverse effects on cardiac smooth muscle cells. The mechanisms of these effects are not fully understood. One possible mechanism involves oxidative stress. Therefore, we investigated the effect of aldosterone and NaCl on rat aortic smooth muscle cells by measuring the activity and expression of the key enzyme of the pentose-phosphate pathway, glucose-6-phosphate-dehydrogenase (G6PD), which provides NADPH to protect the cells from oxidative damage. The formation of reactive oxygen species (ROS) and the role of cAMP were determined. Additionally, the release of the extracellular matrix proteins collagen III and fibronectin have been studied. Our results show that after 48 hours exposure to 10 nM aldosterone or 15 mM NaCl or both the overall protein content decreased by roughly 20%. The formation of ROS was clearly increased after aldosterone exposure (5–6-fold). The enzyme activity of G6PD was slightly decreased when cells were exposed to aldosterone or NaCl or both. Interestingly, the level of G6PD mRNA determined by RT-PCR did not reflect this decrease of activity: after 48 hours, G6PD mRNA levels were significantly increased. G6PD mRNA was reduced after 6 hours aldosterone, a point of time at which the enzyme activity of G6PD was unchanged. Stimulation of the adenylyl cyclase by forskolin led to an increase of G6PD mRNA level but not so to its activity which shows that the PKA-mediated inactivation of G6PD either plays no role or was outbalanced by an increased synthesis rate. In the presence of aldosterone, the forskolin-induced raise of G6PD mRNA-level was significantly reduced but the enzyme activity remained nearly unchanged. The release of collagen III and fibronectin was considerably reduced when aldosterone and/or NaCl were administered. We conclude that 48 hours exposure of smooth muscle cells to aldosterone and/or NaCl leads to a decrease in protein content, an alteration of extracellular matrix, an increase in ROS and to a decrease in G6PD activity. These changes may therefore at least in part responsible for the observed damage of vascular smooth muscle cells after aldosterone and/or NaCl exposure.