Interstitial cells of Cajal (ICC) in the urethra are considered to be putative pacemaker cells which are involved in the generation of spontaneous myogenic tone (Sergeant et al., 2000). Urethral ICC are spontaneously active and the frequency of this activity is modulated by several neurotransmitters, including noradrenaline and nitric oxide, (Sergeant et al., 2002 & 2006). ATP is also considered to be an important neurotransmitter in the urethra, therefore experiments were performed to test if spontaneous activity in urethral ICC was also regulated by ATP. New Zealand white rabbits were humanely killed and ICC were freshly dispersed from the urethra as described previously (Sergeant et al., 2000). ICC were then plated and loaded with Fluo-4 AM for study of cytosolic Ca2+ signals using a spinning disk confocal microscope. STICs were recorded at -60 mV using the amphotericin B perforated patch technique. ATP (10 µM) increased the frequency of STICs in urethral ICC from 9 ± 1.6 to 26 ± 3.7 min-1 (p<0.05). These effects were mimicked by the P2Y receptor agonist 2-methylthio ADP (2-MeSADP, 1 µM), which increased STIC frequency from 7.25 ± 0.5 to 26.25 ± 3.2 min-1, n=8 (p<0.05). The broad spectrum purinergic receptor antagonist suramin (100 uM) reduced the mean frequency of ATP evoked inward currents from 17.9 ± 2.8 to 1.3 ± 0.66 min-1 (n = 9, p<0.05) and those evoked by 2-MeSADP from 31 ± 6.1 to 4.2 ± 2.1 min-1 (n = 6, p<0.05). Similar results were achieved with the selective P2Y1 receptor antagonist, MRS2500 (100 nM) which reduced the mean frequency of ATP evoked inward currents from to 17 ± 4.5 min-1 to 6 ± 2.5 min-1 (n= 8, p<0.05) and 2-MeSADP induced currents from 25.8 ± 4.3 to 7 ± 1.8 min-1 (n= 6, p<0.05). Johnston et al., (2005) showed that STICs in urethral ICC are associated with global Ca2+ oscillations therefore we investigated if this activity was also modulated by ATP and 2-MeSADP. Application of ATP (10 µM) significantly increased the frequency of Ca2+ waves from 9 ± 1.4 to 29.1 ± 4.5 min-1 (n = 14, p<0.05) and 2-MeSADP (1 µM) caused an increase from 7.3 ± 1.6 to 17.1 ± 2.8 min-1 (n=10, p<0.05). The frequency of the ATP induced Ca2+ oscillations was reduced from 27 ± 8.7 min-1 to 6.6 ± 2.1 min-1 by suramin (n=7, p<0.05) and similar results were achieved with MRS2500, which decreased the mean frequency of ATP induced Ca2+ oscillations from 28.1 ± 4.2 to 10.3 ± 3.9 min-1 (n= 6, p<0.05). These data demonstrate that spontaneous activity in urethral ICC is modulated by ATP and that this effect is likely to be mediated by P2Y receptors.