Urinary bladder smooth muscle contraction is triggered by parasympathetic nerves, which release acetylcholine and ATP that bind to muscarinic and purinergic receptors, respectively. The contributions of these receptors to myosin regulatory light chain (RLC) phosphorylation and contraction may be distinct. Both receptors mediate Ca2+ influx whereas muscarinic receptors may also recruit Ca2+-sensitization mechanisms. Using transgenic mice expressing CaM-sensor myosin light chain kinase (MLCK) in smooth muscles, effects of atropine or suramin/αβ-meATP on neurally stimulated force, MLCK activation, and phosphorylation of RLC, myosin light chain phosphatase (MLCP) targeting subunit MYPT1 and MLCP inhibitor protein CPI-17 were examined. Measurements of MLCK activation showed the purinergic component dominant in the initial phase of contraction and the muscarinic component prominent in the sustained phase. Phosphorylation of RLC and CPI-17 was increased upon stimulation with maximal responses at 20 Hz for 10 sec; responses were blunted by atropine and suramin/αβ-meATP. Stimulation did not evoke MYPT1 phosphorylation at T696 or T850. Thus, purinergic Ca2+ signals provide the initial activation of MLCK with cholinergic receptors supporting sustained responses. Further, both receptors recruit CPI-17-mediated MLCP inhibition to increase RLC phosphorylation, and thus contraction. HL26043