Although nicotinic acetylcholine receptors in both the central and peripheral nervous systems play a prominent role in the control of urinary bladder function, little is known regarding expression or function of nicotinic receptors in the bladder epithelium, or urothelium. Nicotinic receptors have been described in epithelial cells lining the upper GI tract, respiratory tract, and the skin. Thus, the present study examined the expression and functionality of nicotinic receptors in the urothelium, as well as the effects of stimulation of nicotinic receptors on the micturition reflex. mRNA for the 3, 5, 7, 3, and 4 nicotinic subunits was identified in rat urothelial cells using RT-PCR. Western blot also confirmed urothelial expression of the 3 and 7 subunits. Application of nicotine (50nM) to cultured rat urothelial cells elicited an increase in [Ca⁺²]_i, indicating that at least some of the subunits form functional channels. These effects were blocked by the application of the nicotinic antagonist hexamethonium. During in vivo bladder cystometrograms in urethane anesthetized rats, intravesical administration of nicotine, choline or the antagonists methyllycaconitine citrate and hexamethonium elicited changes in voiding parameters. Intravesical nicotine (50nM, 1µM) increased the intercontraction interval (ICI). Intravesical choline (1-100µM) also affected bladder reflexes similarly, suggesting that 7 nicotinic receptors mediate this effect. Intravesical administration of hexamethonium (1-100µM) potentiated the nicotine-induced changes in bladder reflexes. Methyllycaconitine citrate, a specific 7-receptor antagonist, prevented nicotine-, choline- and hexamethonium-induced bladder inhibition. These results are the first indication that stimulation of non-neuronal nicotinic receptors in the bladder can affect micturition.