Overactive bladder (OAB) is accompanied by abnormal muscle contraction and increased ATP release from the mucosa. An important common factor is an increase of intracellular Ca2+, which in turn activates a central intracellular signalling pathway triggered by the Ca2+-calmodulin-dependent protein phosphatase, calcineurin (Cn). An increase of Cn activity is associated with the development of an overactive bladder phenotype, that may represent a direct action or the secondary activation of other phosphatases such as PP1 and PP2A. The role of these phosphatases may be examined by the use of inhibitors such as cyclosporin (CsA for Cn) or okadaic acid (for PP1/PP2A). To date, the physiological role of Cn-dependent pathways in bladder contractions is unclear. The objectives of this study were: 1) to examine the effects of CsA or okadaic acid on spontaneous and carbachol-evoked, as well as nerve-mediated contractions and 2) the effect of CsA on mucosal-mediated ATP release upon muscle stretch. Muscle strips were dissected from rabbit urinary bladders, and superfused with normal Tyrode’s solution in horizontal organ baths. Isometric contractions were measured from preparations with the mucosa attached or denuded. Contractions were evoked in the absence and presence of CsA by either exposure to the muscarinic receptor agonist, carbachol or electrical field stimulation (20Hz). ATP release from mucosa-intact preparations after a rapid stretch (20% resting length) was measured from superfusate samples using a luciferin-luciferase assay. All data are mean±S.D., differences between data sets were tested with Student’s t-test and the null hypothesis rejected at p<0.05. Spontaneous contractions were suppressed by CsA at 1, 5 and 10 μM and also by 0.1 μM okadaic acid (CsA 36.2±12.2 % control; n=6; okadaic acid 40.2±23.1% control; n=4; p<0.01). Contractures evoked by 0.1 μM carbachol were unaffected by 1μM CsA but significantly attenuated at 5 and 10 μM (74.8±5.9 and 75.9±4.3% control respectively; n=5; p<0.05). Okadaic acid also reduced carbachol contractions (73.6± 11.8 % control; n=4; p<0.05). CsA also attenuated contractions from denuded strips evoked by electrical field stimulation, but only to 95±4% control, n=4, p<0.05). Finally, CsA reduced stretch-evoked ATP release from mucosa-intact preparations (73.8±4.4 % control; n=10, p<0.05). Inhibition of intracellular phosphatase activity supressed both spontaneous and carbachol-induced contractions, but had a relatively small effect on nerve-mediated contractions: the effect of CsA was more potent on spontaneous activity. CsA also attenuated stretch-induced ATP release, which is a regulator of spontaneous activity. The data suggest that intracellular phosphatases are key regulators of several steps in the pathways that generate contraction in bladder smooth muscle.