Myogenic tone in urethra smooth muscle is regulated by several neurotransmitters, including noradrenaline, acetylcholine and nitric oxide (Brading et al., 1999). ATP is also considered to be an important neurotransmitter in the urethra (Pinna et al., 1996), however its effects have not yet been fully characterised and therefore it’s physiological role remains unclear. The purpose of the present study was to characterise the effects of ATP on the contractile activity of rabbit urethral smooth muscle. New Zealand white rabbits were humanely killed and mechanical recordings were made from strips of proximal urethra (8 x 1 x 1 mm) using a multi channel Myobath system. Muscle strips were perfused with warmed Krebs solution bubbled with 95% O2-5% CO2 which contained atropine (1 µM), phentolamine (1 µM) and NG-Nitro-L-arginine (NO-ARG, 100 µM). Strips were adjusted to a tension of 2-4 mN and allowed to equilibrate for 50 min before experimentation began. Exogenous application of ATP (10 µM) induced robust contractions of the muscle strips. These responses typically consisted of an increase in tone, superimposed upon which were transient phasic contractions that varied in frequency and amplitude. The contractile effects were quantified by measuring the total area under the trace (mN.s). In 45 muscle strips ATP increased the tone from 43.6 ± 20.8 to 163 ± 40.1 mN.s (p<0.05). The ATP induced contractions were inhibited by the broad spectrum purinergic inhibitor suramin (100 µM) from 95 ± 18 to 37 ± 16 mN.s (n=17, p<0.05) but were not affected by the P2X receptor selective antagonist NF279 (1 µM, 99 ± 31 mN.s under control conditions versus 101 ± 21 mN.s in its presence, p>0.05). In contrast, the ATP effects were greatly attenuated by the P2Y1 receptor antagonist MRS2500 (100 nM) and were mimicked by the P2Y receptor agonist, 2-methylthio ADP (2-MeSADP). For example, in 18 muscle strips the mean ATP evoked contraction was reduced from 118 ± 23 to 65 ± 15 mN.s by MRS2500 (p<0.05) whereas 2-MeSADP (1 µM), induced a contraction measuring 372 ± 108 mN.s compared to 153 ± 44 mN.s produced by ATP (10 µM) in paired experiments (p<0.05, n = 14). These results suggest that ATP could act as an excitatory neurotransmitter and that these effects are likely to be mediated via P2Y receptors.