Urethral smooth muscle generates spontaneous electrical slow waves that are thought to underpin contraction of this tissue (Hashitani et al., 1996). Several studies suggest that this activity is initiated by activation of Ca2+-activated Cl- channels (CACCs) in specialised pacemaker cells known as interstitial cells of Cajal (ICC, Sergeant et al., 2000). However, the exact contribution of CACCs to myogenic tone in the urethra has not yet been ascertained, in part, because their molecular identity has not been determined in this tissue. The purpose of the present investigation was to determine if the novel Ca2+-activated Cl- channel, TMEM16A encoded CACCs in rabbit urethral smooth muscle (RUSM). The patch clamp technique was used to record Ca2+-activated Cl- currents (IClCa) in freshly isolated ICC from the rabbit urethra and stably expressed TMEM16A currents in HEK 293 cells. In addition, tension recordings were made from strips of RUSM using a WPI Myobath system. All experiments were approved by the Dundalk Institute of Technology animal care and use committee. Data are presented as mean ± SEM. Current-voltage relationships were obtained from HEK cells stably expressing human TMEM16A, cloned from the testes. These currents were inhibited by the novel TMEM16A inhibitor, TinhA01 (10 µM) across the voltage range and in 5 cells TinhA01 reduced the mean current evoked by a step to +100 mV from 1.27 ± 0.23 to 0.3 ± 0.06 nA (p<0.05, paired t test). Similarly, TinhA01 (30 µM) reduced the peak IClCa evoked by a step to +50 mV in native ICC from a mean of 797 ± 200 pA to 253 ± 69 pA (n=5, p<0.05, paired t test). Urethral ICC develop spontaneous transient inward currents (STICs) when voltage clamped at -60 mV (Sergeant et al., 2000). The amplitude of these events were also inhibited by TinhA01, from a mean of -647 ± 152 pA under control conditions to -30 ± 25 pA in the presence of the drug (n=6, p<0.05, paired t test). Next we investigated the effect of TinhA01 on contractions of RUSM evoked by electrical field stimulation at frequencies of 2, 4 and 8Hz (pulse width 0.3 ms, nominal amplitude 20 V). TinhA01 significantly reduced the peak contraction evoked during 4Hz stimulation from a mean value of 2 ± 0.4 mN under control conditions to 0.4 ± 0.1 mN in its presence (n=9, p<0.05). In addition, TinhA01 also decreased spontaneous myogenic tone in strips of RUSM by 0.4 ± 0.07 mN. Finally, contractions of RUSM evoked by exogenous application of phenylephrine (1 µM) were inhibited by TinhA01 from a mean value of 8.9 ± 1.9 to 7 ± 1.4 mN (n=8, p<0.05). Taken together these data suggest that TMEM16A channels contribute to IClCa in urethral ICC and that these channels are crucially involved in the generation and modulation of urethral tone.