Protein kinase C (PKC) stimulation of NAD(P)H oxidases (Nox) is an important component of multiple vascular disease processes, however, the relationship between oxidase activation and the regulation vascular smooth muscle contraction by PKC remains poorly understood. Therefore, we examined the signaling cascade of PKC-elicited Nox activation and the role of superoxide and hydrogen peroxide in mediating PKC induced vascular contraction. Endothelium-denuded bovine coronary arteries showed a PKC-dependent basal production of lucigenin (5 mM)-detected Nox oxidase-derived superoxide, which was stimulated 4-fold by PKC activation with 10 µM phorbol 12,13-dibutyrate (PDBu). PDBu appeared to increase superoxide generation by Nox2 through both p47^phox and peroxide-dependent Src activation mechanisms based on the actions of inhibitors, properties of Src phosphorylation, and the loss of responses in aorta from mice deficient in Nox2 and p47^phox. The actions of inhibitors of contractile regulating mechanisms, scavengers of superoxide and peroxide, and responses in knockout mouse aortas, suggest that a major component of the contraction elicited by PDBu appeared to be mediated through peroxide derived from Nox2 activation stimulating force generation through Rho kinase and calmodulin kinase-II mechanisms. Superoxide generated by PDBu also attenuated relaxation to nitroglycerin. Peroxide-derived from Nox2 activation by PKC appeared to be a major contributor to the thromboxane A2 receptor agonist U46619 (100 nM)-elicited contraction of coronary arteries. Thus, a p47^phox and Src kinase activation of peroxide production by Nox2 appears to be an important contributor to vascular contractile mechanisms mediated through activation of PKC.