Ca²⁺/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to [Ca²⁺]_i. We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca²⁺]_i with carbachol compared to KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was non-linear such that at higher concentrations of carbachol, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of MYPT1 and CPI-17, respectively. Carbachol treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced carbachol-dependent force, RLC phosphorylation and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C but not Y27632 also reduced carbachol-induced phosphorylation of CPI-17. Carbachol concentration responses showed phosphorylation of CPI-17 was more sensitive than MYPT1. Thus, the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.