It has been previously determined that the epithelial lining of the urinary bladder, or urothelium, expresses two subtypes of nicotinic acetylcholine receptors (nAChRs) that mediate distinct physiological effects in vivo. These effects include inhibition of bladder reflexes through α7 receptors and an excitation of bladder reflexes through α3-containing (α3*) receptors. It is believed that urothelial receptors mediate their effects through modulating the release of neurotransmitters such as ATP that subsequently influence bladder afferent nerve excitability. Therefore, we examined the distribution of nAChRs in the urothelium, as well as their ability to influence the release of the neurotransmitter ATP. Immunofluorescent staining of both whole bladder tissue and primary urothelial cultures from the rat demonstrated that the urothelium contains both α3* and α7 receptors. In primary urothelial cultures, α7 stimulation with choline (10 μm to 1 mm) caused a decrease in basal ATP release while α3* stimulation with cytisine (1–100 μm) caused a concentration-dependent, biphasic response, with low concentrations (1–10 μm) inhibiting release and higher concentrations (50–100 μm) increasing release. These responses were mirrored in an in vitro, whole bladder preparation. In vivo, excitation of bladder reflexes in response to intravesical cytisine (100 μm) is blocked by systemic administration of the purinergic antagonist PPADS (1 or 3 μg kg−1). We also examined how each receptor subtype influenced intracellular Ca2+ levels in cultured urothelial cells. nAChR stimulation increased [Ca2+]i through distinct mechanisms: α7 through a ryanodine-sensitive intracellular mechanism and α3* through extracellular influx. In addition, our findings suggest interactions between nAChR subtypes whereby activation of α7 receptors inhibited the response to a subsequent activation of α3* receptors, preventing the increase in [Ca2+]i previously observed. This inhibitory effect appears to be mediated through protein kinase A- or protein kinase C-mediated pathways.