We have shown earlier a requirement for Ca(2+) and calmodulin (CaM) in the H(2)O(2)-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key mediators of growth-promoting, proliferative, and hypertrophic responses in vascular smooth muscle cells (VSMC). Because the effect of CaM is mediated through CaM-dependent protein kinase II (CaMKII), we have investigated here the potential role of CaMKII in H(2)O(2)-induced ERK1/2 and PKB phosphorylation by using pharmacological inhibitors of CaM and CaMKII, a CaMKII inhibitor peptide, and siRNA knockdown strategies for CaMKII alpha. Calmidazolium and W-7, antagonists of CaM, as well as KN-93, a specific inhibitor of CaMKII, attenuated H(2)O(2)-induced responses of ERK1/2 and PKB phosphorylation in a dose-dependent fashion. Similar to H(2)O(2), calmidazolium and KN-93 also exhibited an inhibitory effect on glucose/glucose oxidase-induced phosphorylation of ERK1/2 and PKB in these cells. Transfection of VSMC with CaMKII autoinhibitory peptide corresponding to the autoinhibitory domain (aa 281-309) of CaMKII and with siRNA of CaMKII alpha attenuated the H(2)O(2)-induced phosphorylation of ERK1/2 and PKB. In addition, calmidazolium and KN-93 blocked H(2)O(2)-induced Pyk2 and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation. Moreover, treatment of VSMC with CaMKII alpha siRNA abolished the H(2)O(2)-induced IGF-1R phosphorylation. H(2)O(2) treatment also induced Thr(286) phosphorylation of CaMKII, which was inhibited by both calmidazolium and KN-93. These results demonstrate that CaMKII plays a critical upstream role in mediating the effects of H(2)O(2) on ERK1/2, PKB, and IGF-1R phosphorylation.