Vascular calcification is enhanced with aging and in atherosclerosis, with functional consequences in hemodynamics and plaque rupture. Mechanisms underlying such calcification remain unclear. Since redox processes are involved in the genesis of most vascular diseases, we hypothesized that such processes may contribute to vascular and aortic valve calcification. We showed previously in a rabbit model that hypercholesterolemia + vitamin D₃ supplementation triggered massive vascular and aortic valve calcification and that Tempol, a nitroxide with antioxidant properties, did not reduce, but slightly increased this process. In the present study, we aimed to validate an in vitro model of vascular smooth muscle cell (VSMC) calcification and to investigate in this process the occurrence of oxidative stress and effects of antioxidants. VSMC were kept for 14 days in calcifying media containing CaCl₂ (6mM) plus ß-glycerophosphate (10mM) and/or vitamin D₃ (0.1uM). We assessed the expression of factors involved in VSMC dedifferentiation into calcifying vascular cells, such as Osteopontin (OPN), Osteoprotegerin (OPG) and Cbfa-1. RT-PCR analysis showed enhanced expression of OPN mRNA in the presence of ß-glycerophosphate, especially when it was added together with vitamin D₃ and tempol (0.1mM). Cbfa-1 mRNA, which was absent in intact VSMC, was induced by ß-glycerophosphate and vitamin D₃ , but also by tempol incubation. Western Blotting analysis showed enhanced expression of OPN and OPG after co-incubation with tempol both in the presence of CaCl₂ alone and particularly with ß-glycerophosphate plus vitamin D₃. Assessment of NADPH oxidase activity in VSMC membrane fraction (lucigenin luminescence) showed increased activity when ß-glycerophosphate or  vitamin D₃ were added separatedly, which was not observed with tempol incubation. Thus, our results suggest that redox processes and NADPH oxidase activity are involved in dedifferentiation of VSMC into calcifying vascular cells in a way that at least the antioxidant tempol can potentiate this process. (Supported by FAPESP and CNPq Milênio Redoxoma).