Although urethral tone is spontaneous, it can be modulated by inhibitory nitrergic and excitatory noradrenergic and cholinergic nerves (Thornbury et al., 1992). The purpose of this study was to identify which acetylcholine receptor subtype was responsible for increasing tone in the proximal rabbit urethra and examine how their stimulation affected spontaneous electrical activity. Rabbits were humanely killed with pentobarbitone (I.V.) and the proximal 3 cm of the urethra was removed. The urothelium was removed before the preparation was pinned out on a silicon rubber base and superfused with Krebs solution at 35-37oC. For isometric tension recording experiments, circular strips of urethra were adjusted to a resting tension of 5 mN and superfused with Krebs solution at 35-37oC containing 100 μM NO-ARG and 1_μM guanethidine. When contractions were elicited by transmural nerve stimulation (0.5 Hz – 4 Hz, 50 V, 0.3 ms pulse width, 60 s duration) via electrodes placed at either end of the organ bath, frequency dependent increases in tone were observed and these were blocked by tetrodotoxin (1 μM, n=4). The peak amplitude of the neurogenic contractions evoked by stimulating at 4 Hz was significantly reduced from 19.1±4.6 mN to 7.2±2.3 mN (p<0.05, mean±SEM, paired t-test) by atropine (1 μM), suggesting that muscarinic receptors were involved in this response. Methoctramine (1 μM, M2 blocker) had little effect on these responses (n=6). However, the M1/M3 muscarinic receptor antagonist 4-DAMP (100 nM) significantly reduced the neurogenic contractions evoked by 2 Hz and 4 Hz from 1.2±0.3 and 2.3±0.4 mN to 0.4±0.2 and 1.0±0.4 mN (p<0.05, n=6) respectively, suggesting that acetylcholine mediates its effects on tone via activation of M1 or M3 receptors. When the proximal urethra was impaled with sharp microelectrodes, regular spontaneous slow waves which had spikes superimposed upon a plateau were recorded as demonstrated previously (Bradley et al., 2004). Carbachol increased the frequency of the slow waves from 3.1±0.3 min-1 (n=9) to 3.3±0.8 (n=3), 5.9±0.5 (n=6), 6.8±0.8 (n=4), 12.3±2.2 (n=4) min-1 in response to 10 nM, 100 nM, 300 nM and 1μM carbachol respectively. To examine if this effect was via stimulation of M1/M3 receptors, we examined the effects of 300 nM carbachol on electrical activity before and during incubation of the tissue with 4-DAMP (100 nM). In five experiments, carbachol (300 nM) increased slow wave frequency from 4.1±0.6 min—1 to 7.1±1.6 min-1 (p<0.05). In the presence of 4-DAMP, frequency was 3.8 ± 0.5 min-1 compared to 4.0±0.6 min-1 during 4-DAMP and carbachol. These data suggest that muscarinic agonists increase tone by modulating the frequency of slow waves in the proximal urethra via activation of M1/M3 receptors.