TRP and ASIC channels are molecular detectors of chemical, mechanical, thermal and nociceptive stimuli in sensory neurons. They have been identified in the urothelium, a tissue considered part of bladder sensory pathways, where they might play a role in bladder function. This study investigated functional properties of TRP and ASIC channels in cultured urothelial cells from the rat using patch clamp and fura-2 Ca²⁺ imaging techniques. The TRPV4 agonist 4PDD (1-5 µM) and the TRPA1/TRPM8 agonist icilin (50-100 µM) elicited transient currents in a high percentage of cells (>70%). 4PDD responses were suppressed by the TRPV4 antagonist HC-010961 (10 µM). The TRPV1 agonist capsaicin (1-100 µM) and the TRPA1/TRPM8 agonist menthol (5-200 µM) elicited transient currents in a moderate percentage of cells (~25%). All of these agonists increased intracellular calcium concentration ([Ca²⁺]_i). Most cells responded to more than one TRP agonist (e.g. capsaicin and 4PDD) indicating co-expression of different TRP channels. In the presence of the TRPV1 antagonist capsazepine (10 µM), changes in pH induced by HCl elicited ionic currents (pH 5.5) and increased [Ca²⁺]_i (pH 6.5) in ~50% of cells. Changes in pH using acetic acid (pH 5.5) elicited biphasic-like currents. Responses induced by acid were sensitive to amiloride (10 µM). In summary, urothelial cells express multiple TRP and ASIC channels whose activation elicits ionic currents and Ca²⁺ influx. These 'neuron-like' properties might be involved in transmitter release, such as ATP, that can act on afferent nerves or smooth muscle to modulate their responses to different stimuli.