We previously demonstrated that excitatory agonists such as noradrenaline (NA) and membrane depolarization induce Ca2+-dependent activation of Rho GTPase in vascular smooth muscle (VSM) cells, resulting in inhibition of myosin phosphatase (MP) through the mechanisms involving Rho kinase-mediated phosphorylation of its regulatory subunit MYPT1/MBS. We found that phosphoinositide 3-kinase class IIα (PI3K-C2α) plays an essential role in NA-induced Rho activation and contraction in differentiated VSM primary cultured-cells. In the present study we show that ionomycin, a Ca2+ ionophore, induced contraction with stimulated phosphorylation of MYPT1. Ionomycin-induced MYPT1 phosphorylation and contraction was inhibited by the Rho kinase inhibitor Y-27632. Silencing PI3K-C2α, but not PI3K p110α, expression by small interfering RNA (siRNA) in differentiated VSM cells inhibited ionomycin-induced phosphorylation of MYPT1, consequent reinforcement of 20-kDa myosin light chain (MLC) phosphorylation and contraction. Consistent with this, the PI3K inhibitors Wortmannin and LY294002 inhibited both MLC phosphorylation and contraction. These findings indicate an essential role of PI3K-C2α in Ca2+-dependent, Rho/Rho kinase-mediated negative control of MP and VSM contraction. [J Physiol Sci. 2006;56 Suppl:S73]