Protein kinase C (PKC) activation by a phorbol ester increases myosin light chain (MLC(20)) phosphorylation through inhibition of MLC phosphatase (MLCP) and enhances contraction of vascular smooth muscle. We investigated whether Rho kinase, which is known to inhibit MLCP, is involved in the MLC(20) phosphorylation caused by a phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), in rabbit aortas. DPB (1 microM) increased MLC(20) phosphorylation and tension. The Rho kinase inhibitor fasudil (10 microM) inhibited the DPB-induced contraction and decreased the MLC(20) phosphorylation at Ser19, a site phosphorylated by MLC kinase, although it did not affect the phosphorylation of total MLC(20). Rinsing a 65.4 mM KCl-contracted aorta with Ca(2+)-free, EGTA solution caused rapid dephosphorylation of MLC(20) and relaxation. When DPB was present in the rinsing solution, the MLC(20) dephosphorylation and the relaxation were inhibited. In this protocol, Ro31-8220 (10 microM), a PKC inhibitor, suppressed the phosphorylation of total MLC(20) and Ser19 induced by DPB. Fasudil also inhibited the Ser19 phosphorylation to a degree similar to Ro31-8220 and accelerated relaxation, which was less than the relaxation caused by Ro31-8220. The phospholipase A(2) inhibitor ONO-RS-082 (5 microM) inhibited the DPB-induced Ser19 phosphorylation but only transiently decreased the tension, suggesting the involvement of arachidonic acid in the phosphorylation and the existence of a MLC(20) phosphorylation-independent mechanism. When fasudil was combined with ONO-RS-082, fasudil exerted additional inhibition of the tension without further inhibition of the Ser19 phosphorylation. DPB phosphorylated the 130 kDa myosin binding subunit (MBS) of MLCP and fasudil inhibited the phosphorylation. These data suggest that the inhibition by fasudil of DPB-induced contraction and phosphorylation of MLC(20) at the MLC kinase-targeted site is a result of inhibition of Rho kinase. Thus, the PKC-dependent Ca(2+)-sensitization of vascular smooth muscle involves Rho kinase. A MLC(20) phosphorylation-independent mechanism is also involved in the Ca(2+)-sensitization.