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Tyrosine kinases, typically associated with growth-signaling pathways, also play a role in Ang II-stimulated vascular contraction. However the specific kinases involved are unclear. We hypothesize here that c-Src, a non-receptor tyrosine kinase, is an important upstream regulator of vascular smooth muscle cell (VSMC) Ca2+ signaling and associated vascular contraction induced by Ang II.Cultured VSMCs from resistance arteries of healthy subjects were studied. Human VSMCs electroporated with anti-c-Src antibody and c-Src-deficient VSMCs from small arteries of c-Src knockout mice (Src-/-mVSMCs) were also investigated. Intracellular free Ca2+ concentration ([Ca2+]i), c-Src activity and IP3 production were measured by fura 2, immunoblot and radioimmunoassay respectively. Contraction was examined in intact rat small arteries.Ang II rapidly increased VSMC c-Src activity, with peak responses obtained at 1 min. Ang II induced a biphasic [Ca2+]i response (Emax= 636 ± 123 nmol/l). The initial [Ca2+]i transient, mediated primarily by Ca2+mobilization, was dose-dependently attenuated by the selective Src inhibitor, PP2, but not by PP3 (inactive analogue). Ang II-elicited [Ca2+]i responses were blunted in cells electroporated with anti-c-Src antibodies and in c-Src–/–mVSMCs. Src inhibition decreased Ang II-induced generation of IP3 in human VSMCs. Ang II dose-dependently increased vascular contraction (Emax= 40 ± 6.5%). These responses were attenuated by PP2 (Emax= 7.8 ± 0.08%) but not by PP3 (Emax= 35 ± 4.5%).Our findings identify c-Src as an important regulator of VSMC [Ca2+]i signaling and implicate a novel contractile role for this non-receptor tyrosine kinase in Ang II-stimulated vascular smooth muscle.