The initiation of contractile force in arterial smooth muscle (SM) is believed to be regulated by the intracellular Ca2+ concentration and SM myosin type II phosphorylation. We tested the hypothesis that SM myosin type II operates as a molecular motor protein in electromechanical, but not in protein kinase C (PKC)-induced, contraction of small resistance-sized cerebral arteries. We utilized a SM type II myosin heavy chain (MHC) knockout mouse model and measured arterial wall Ca2+ concentration ([Ca2+](i)) and the diameter of pressurized cerebral arteries (30-100 microm) by means of digital fluorescence video imaging. Intravasal pressure elevation caused a graded [Ca2+](i) increase and constricted cerebral arteries of neonatal wild-type mice by 20-30%. In contrast, intravasal pressure elevation caused a graded increase of [Ca2+](i) without constriction in (-/-) MHC-deficient arteries. KCl (60 mM) induced a further [Ca2+](i) increase but failed to induce vasoconstriction of (-/-) MHC-deficient cerebral arteries. Activation of PKC by phorbol ester (phorbol 12-myristate 13-acetate, 100 nM) induced a strong, sustained constriction of (-/-) MHC-deficient cerebral arteries without changing [Ca2+](i). These results demonstrate a major role for SM type II myosin in the development of myogenic tone and Ca2+ -dependent constriction of resistance-sized cerebral arteries. In contrast, the sustained contractile response did not depend on myosin and intracellular Ca2+ but instead depended on PKC. We suggest that SM myosin type II operates as a molecular motor protein in the development of myogenic tone but not in pharmacomechanical coupling by PKC in cerebral arteries. Thus PKC-dependent phosphorylation of cytoskeletal proteins may be responsible for sustained contraction in vascular SM.