Sensory neurons represent an attractive target for pharmacological treatment of various bladder disorders. However the properties of major classes of mechano-sensory neurons projecting to the bladder have not been systematically established. An in vitro bladder preparation was used to examine the effects of a range of mechanical stimuli (stretch, von Frey hair stroking and focal compression of receptive fields) and chemical stimuli (1 mm alpha,beta-methylene ATP, hypertonic solutions (500 mm NaCl) and 3 microm capsaicin) during electrophysiological recordings from guinea pig bladder afferents. Four functionally distinct populations of bladder sensory neurons were distinguished by these stimuli. The first class, muscle mechanoreceptors, were activated by stretch but not by mucosal stroking with light (0.05-0.1 mN) von Frey hairs or by hypertonic saline, alpha,beta-methylene ATP or capsaicin. Removal of the urothelium did not affect their stretch-induced firing. The second class, muscle-mucosal mechanoreceptors, were activated by both stretch and mucosal stroking with light von Frey hairs or by hypertonic saline and by alpha,beta-methylene ATP, but not by capsaicin. Removal of the urothelium reduced their stretch- and stroking-induced firing. The third class, mucosal high-responding mechanoreceptors, were stretch-insensitive but could be activated by mucosal stroking with light von Frey hairs or by hypertonic saline, alpha,beta-methylene ATP and capsaicin. Stroking-induced firing was significantly reduced by removal of the urothelium. The fourth class, mucosal low-responding mechanoreceptors, were stretch insensitive but could be weakly activated by mucosal stroking with light von Frey hairs but not by hypertonic saline, alpha,beta-methylene ATP or capsaicin. Removal of the urothelium reduced mucosal stroking-induced firing. All four populations of afferents conducted in the C-fibre range and showed class-dependent differences in spike amplitude and duration. At least four functional classes of bladder mechanoreceptors can be readily distinguished by different mechanisms of activation and are likely to transmit different types of information to the central nervous system.