We tested the hypothesis that endothelial nitric oxide synthase (eNOS)-derived NO modulates rho-kinase-mediated vascular contraction. Because HMG-CoA-reductase inhibition can both upregulate eNOS expression and inhibit rhoA/rho-kinase function, a second hypothesis tested was that statin treatment modulates rho-kinase-mediated contraction, and that this can occur independently of eNOS. Contractile responses to the receptor-dependent agonists, serotonin and phenylephrine, but not to the receptor-independent agent, potassium chloride (KCl), were greater in aortic rings from eNOS-null (eNOS^-/-) versus wild-type (eNOS^+/+) mice. Similarly enhanced responses were seen in eNOS^+/+ rings following acute NOS inhibition. The rho-kinase inhibitor, Y-27632, abolished or profoundly attenuated responses to receptor agonists in both eNOS^+/+ and eNOS^-/- rings, but responses in eNOS^+/+ were more sensitive to Y-27632. Mevastatin treatment (20 mg/kg per d sc, 14 d) reduced responses to serotonin and phenylephrine in female mice of both strains. KCl-induced contractions were slightly smaller in eNOS^+/+-derived aortic rings only. Levels of plasma cholesterol, and aortic expression of rhoA and rho-kinase, did not differ between groups. Thus, eNOS-derived NO suppresses rhoA/rho-kinase-mediated vascular contraction. Moreover, a similar suppressive effect on rho-kinase-mediated vasoconstriction by statin therapy occurs independently of effects on eNOS or plasma cholesterol.