Several hypertensive states are associated with resistance to insulin-induced glucose disposal and insulin-induced vasodilation. Insulin can inhibit vascular smooth muscle (VSM) contraction at the level of the VSM cell, and resistance to insulin's inhibition of VSM cell contraction may be of pathophysiological importance. To understand the VSM cellular mechanisms by which insulin resistance leads to increased VSM contraction, we sought to determine how insulin inhibits contraction of normal VSM. It has been shown that insulin lowers the contractile agonist-stimulated intracellular Ca²⁺ (Ca²⁺_i) transient in VSM cells. In this study, our goal was to see whether insulin inhibits VSM cell contraction at steps distal to Ca²⁺_i and, if so, to determine whether the mechanism is dependent on nitric oxide synthase (NOS) and cGMP. Primary cultured VSM cells from canine femoral artery were bathed in a physiological concentration of extracellular Ca²⁺ and permeabilized to Ca²⁺ with a Ca²⁺ ionophore, either ionomycin or A-23187. The resultant increase in Ca²⁺_i contracted individual cells, as measured by photomicroscopy. Preincubating cells with 1 nM insulin for 30 min did not affect basal Ca²⁺_i or the ionomycin-induced increase in Ca²⁺_i, as determined by fura 2 fluorescence measurements, but it did inhibit ionomycin- and A-23187-induced contractions by 47 and 51%, respectively (both P < 0.05). In the presence of 1.0 µM ionized Ca²⁺, ionomycin-induced contractions were inhibited by insulin in a dose-dependent manner. In the presence of ionomycin, insulin increased cGMP production by 43% (P < 0.05). 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 µM), a selective inhibitor of guanylate cyclase that blocked cGMP production in these cells, completely blocked the inhibition by insulin of ionomycin-induced contraction. It was found that the cells expressed the inducible isoform of NOS. N^G-monomethyl-L-arginine or N^G-nitro-L-arginine methyl ester (0.1 mM), inhibitors of NOS, did not affect ionomycin-induced contraction but prevented insulin from inhibiting contraction. We conclude that insulin stimulates cGMP production and inhibits VSM contraction in the presence of elevated Ca²⁺_i. This inhibition by insulin of VSM contraction at sites where Ca²⁺_i could not be rate limiting is dependent on NOS and cGMP.