Hyperinsulinemia is a major risk factor for the development of vascular disease. We have reported that insulin increases the motility of vascular smooth muscle cells, via a hydrogen peroxide-mediated mechanism, and that NO attenuates insulin-induced motility, via a cGMP-mediated mechanism. Events downstream of cGMP elevation have not yet been investigated. The aim of our study was to test the hypothesis that antimotogenic effects of NO and cGMP in cultured rat aortic smooth muscle cells are mediated via protein kinase G (PKG), followed by reduction of cytoplasmic Ca²⁺ levels and increased PTP-PEST activity, leading to suppression of agonist-induced elevation of hydrogen peroxide levels and cell motility. Treatment of primary cultures with adenovirus expressing PKG1 mimicked NOinduced inhibition of insulin-elicited hydrogen peroxide elevation and cell motility, whereas treatment with pharmacological PKG inhibitor, Rp-8-Br-cGMPS, rescued the stimulatory effects of insulin that were suppressed by NO donor. Treatment of cells with insulin failed to increase cytoplasmic Ca²⁺ levels, whereas NO donor decreased cytoplasmic Ca²⁺ levels in presence or absence of insulin. Treatment of cells with Ca²⁺ chelator BAPTA mimicked the effects of PKG and NO donor and increased the activity of protein tyrosine phosphatase, PTP-PEST. Finally, treatment with a dominant negative allele of protein tyrosine phosphatase PTP-PEST reversed the inhibitory effect of BAPTA on cell motility and hydrogen peroxide elevation.