—Among the angiotensin II (Ang II)–mediated signal events likely to be important in vascular smooth muscle cells (VSMCs) is activation of extracellular signal–regulated kinases 1 and 2 (ERK1/2). The upstream mediators by which Ang II activates ERK1/2 remain poorly defined. Recently, we showed that Ang II activated c-Src, a nonreceptor kinase, which is a candidate to mediate Ang II signal events. To determine whether c-Src is required for ERK1/2 activation by Ang II, we studied the effects of Src family–selective tyrosine kinase inhibitors on ERK1/2 activation and also studied Ang II–mediated signal events in Src-deficient and Src-overexpressing VSMCs. The tyrosine kinase inhibitors, genistein and CP-188,556, blocked Ang II–mediated ERK1/2 activation in rat VSMCs (rVSMCs). We derived Src-deficient VSMCs from the aortas of c-Src knockout mice (Src^−/− mVSMCs). Basal ERK1/2 activity was lower, and activation of ERK1/2 by Ang II was significantly decreased in Src^−/− mVSMCs compared with wild-type mVSMCs, whereas ERK1/2 protein expression and ERK1/2 activation by phorbol 12-myristate 13-acetate were similar. To examine the role of c-Src further, we overexpressed wild-type or dominant-negative c-Src in rVSMCs using retroviral vectors. ERK1/2 activation by Ang II was significantly increased in rVSMCs that overexpressed c-Src, whereas ERK1/2 activation by Ang II was significantly inhibited in rVSMCs that overexpressed dominant-negative c-Src compared with control rVSMCs. These findings demonstrate that c-Src activation is required for Ang II stimulation of ERK1/2 in VSMCs and suggest an important role for c-Src in Ang II–mediated signal transduction.