The influence of extracellular and intracellular calcium (Ca2+) on smooth muscle contractile activity varies between organs. In response to G protein-coupled receptor (GPCR) stimulation, the urinary bladder detrusor muscle has shown a 70% dependence on extracellular Ca2+, while the urothelium and lamina propria (U&LP) has a 20-50% dependence. However, as this only accounts for partial contractile activity, the contribution of intracellular Ca2+ and Ca2+ sensitization pathways remains unclear. This study assessed the role of intracellular signaling pathways on GPCR-mediated urinary bladder U&LP contraction. Porcine U&LP responses to activation of the muscarinic, histaminergic, 5-hydroxytryptamine (serotonin), neurokinin, prostaglandin, and angiotensin II receptors were assessed with three selective inhibitors of store-released intracellular Ca2+, 2-APB, CPA, and ruthenium red, and three Rho kinase inhibitors, fasudil, Y-27632, and GSK269962. There was no discernible impact on receptor agonist-induced contractions of the U&LP after blocking intracellular Ca2+ pathways, suggesting this tissue is more sensitive to alterations in the availability of extracellular Ca2+. However, an alternative mechanism of action for GPCR-mediated contraction was identified to be the activation of Rho kinase. For example, Y-27632 significantly reduced the GPCR-mediated contractile activity of the U&LP by about 50% (p>0.05, n=8 for all). This suggests that contractile responses of the bladder U&LP do not involve a significant release of Ca2+ from intracellular stores, but that receptor activation causes Ca2+ sensitization via Rho kinase. This study presents a key role for Rho kinase in the urinary bladder, which may provide a novel target for the future pharmaceutical management of bladder contractile disorders.