Background Myosin phosphatase activity is regulated by mechanisms involving the phosphorylation of CPI-17 and MYPT1, primarily based on studies with tonic-type vascular smooth muscles. This study examined how these mechanisms contribute to the regulation of contraction of a phasic-type intestinal smooth muscle. Methods Phosphorylation levels, tension, and Ca2+ sensitization was detected in rat ileal smooth muscle. Key Results In rat ileal smooth muscle, phosphorylation level of CPI-17 at Thr(38) and MYPT1 at Thr(853), but not MYPT1 at Thr(696), were increased with carbachol (1 mu mol L-1) accompanied with muscle contraction. The PKC inhibitor Go6976 (1 mu mol L-1) inhibited the carbachol-induced phosphorylation of CPI-17, whereas the Rho-associated kinase (ROCK) inhibitor, Y-27632 (10 mu mol L-1) inhibited the carbachol-induced phosphorylation of both CPI-17 and MYPT1. Application of Go6976 or Y-27632 alone inhibited the carbachol-induced contraction; however, the combined application of these inhibitors did not inhibit the contraction in an additive manner. In beta-escin-permeabilized ileal strip, treatment with antiphosphorylated antibodies for CPI-17 at Thr(38) and MYPT1 at Thr(853) and Thr(696) alone almost completely abolished the Ca2+ sensitization due to carbachol with GTP. Conclusions & Inferences In conclusion, receptor stimulation increases the Ca2+ sensitivity of contractile elements through CPI-17 phosphorylation via the PKC/ROCK pathways and MYPT1 phosphorylation via the ROCK pathway, when these mechanisms operate cooperatively and/or synchronously in intestinal smooth muscle.