Binding and molecular studies using corporal smooth muscle (CSM) have shown that M2 and M3 muscarinic receptors are present (Traish et al. 1990, 1995). However, the functional subtypes are not known and this was the aim of this study. Guinea-pig CSM strips (~1 mm diameter) were superfused with Tyrode solution (5% CO₂, 24mM NaHCO₃, pH7.4) and attached to an isometric force transducer. Contractions were elicited either by nerve-mediated, electrical field stimulation (EFS: abolished by 1µM tetrodotoxin), or by application of 1.5µM phenylephrine. Relaxation was elicited in pre-contracted strips by 0.3µM carbachol. EFS-induced relaxations were elicited in strips pre-contracted with 15µM phenylephrine. Atropine (1µM), selective muscarinic antagonists pirenzepine (M1), gallamine (M2), 4-DAMP (4-diphenylacetoxy-N-methyl-piperidine methiodide, M3) and L-NAME (100µM) were used to characterise cholinergic and nitric oxide (NO)-mediated relaxation. Contractions are expressed as mN/mm² and muscarinic relaxation as a percentage of the previously-induced contraction. Data are mean ± S.D., differences between means (p<0.05) were examined using Student’s t tests. L-NAME enhanced EFS-induced responses by 60±37% (n=12) at 32Hz stimulation. When strips were pre-treated with atropine, the enhancement by L-NAME was reduced significantly to 21±18% (n=5), implying a component of the NO effect is mediated by a cholinergic pathway. EFS-induced contractions were unaffected by pirenzepine or gallamine (1µM, n=7). However, 4-DAMP (1µM, n=6) enhanced the responses, although there was a large variability of responses (mean percentage increase 46±44%). Carbachol induced a dose-dependent relaxation of the phenylephrine contracture. L-NAME, atropine (n=6) and 4-DAMP (1nM-10µM, n=8) completely reversed this relaxation. Pirenzepine (3nM-100µM, n=8) and gallamine (1nM-100µM, n=8) had no significant effect. EFS also induced a frequency-dependant relaxation of the phenylephrine contracture. The frequency for half-maximal effect (3.4±1.6Hz) was significantly less than that required to induce EFS-contractions (33±2Hz). These relaxations were partially reversed by 4-DAMP (1µM, n=4) at 4Hz stimulation (to 52±16% of control). In turn this reversal was partially offset by subsequent addition of gallamine (1µM, n=4, to 64±9% of control). In guinea-pig CSM, smooth muscle relaxation is mediated by M3 muscarinic receptors via NO release. An M2 muscarinic receptor antagonist (gallamine) modified this response.