Nitric oxide (NO), in addition to its vasodilator action, has also been shown to antagonize the mitogenic and hypertrophic responses of growth factors and vasoactive peptides such as endothelin-1 (ET-1) in vascular smooth muscle cells (VSMCs). However, the mechanism by which NO exerts its anti-mitogenic and anti-hypertrophic effect remains unknown. Therefore, the aim of this study was to determine if NO generation would modify ET-1-induced signaling pathways involved in cellular growth, proliferation and hypertrophy in A-10 VSMC. Treatment of A-10 VSMCs with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP), two NO donors, attenuated the ET-1-enhanced phosphorylation of several key components of growth promoting and hypertrophic signaling pathways such as ERK1/2, PKB and Pyk2. On the other hand, the inhibition of the endogenous NO generation by using N-nitro-L- arginine methyl ester (L-NAME), a NO synthase inhibitor, increased the ET-1-induced phophosphorylation of these signaling components. Since, NO mediates its effect principally through a cyclic GMP/soluble guanylyl cyclase (sGC) pathway, we investigated the role of these molecules in NO action. 8-Br-cGMP, a non-metabolizable and cell permeable analogue of cGMP, exhibited a similar effect to that of SNAP and SNP. Furthermore, oxadiazole quinoxalin (ODQ), an inhibitor of sGC, reversed the inhibitory effect of NO on ET-1-induced responses. SNAP treatment also decreased the protein synthesis induced by ET-1. Taken together, these data demonstrate that NO, in a cGMP-dependent manner, attenuated ET-1-induced phosphorylation of ERK1/2, PKB and Pyk2 and also antagonized the hypertrophic effects of ET-1. It may be suggested that NO-induced generation of cGMP contributes to the inhibition of ET-1-induced mitogenic and hypertrophic responses in VSMCs.