Angiotensin II (Ang II) causes atherosclerosis and hypertension by damaging the vascular structure. One of the major systems causing hypertension is defined as superoxide anion (O2-.), which is known to have a powerful effect on hypertension progression, produced by NAD(P)H oxidase following Ang II stimulation of vascular smooth muscle cells (VSMCs). Our aim in this study has been to determine whether Src phosphorylation in VSMCs precedes NADPH oxidase activation. Spontaneous hypertensive (SHR) and normotensive Wistar-Kyoto rat (WKY) VSMCs were stimulated by Ang II alone and by Ang II after incubation with AT1R, NAD(P)H oxidase, Src, and PKC inhibitors. The cytochrome c reduction method was employed to measure the AT1R-mediated O2-. production by NAD(P)H oxidase. The change in Src phosphorylation was detected by the western blot method after the incubation of VSMCs. Following the Ang II stimulation, Src phosphorylation and O2-. production were found to increase in both groups in comparison with the control group. However, after the Ang II stimulation, Src phosphorylation was reduced with inhibitors other than DPI in both groups. All inhibitors applied after Ang II stimulation suppressed O2- . formation in both groups. The results suggest that Src and protein kinase C phosphorylation precedes NAD(P)H oxidase activation. In this respect, the ability to control Src phosphorylation in the pathway of NAD(P)H oxidase induction by Ang II emerges as important in drug development and treatment of cardiovascular diseases in hypertensive patients.