The pelvic ganglia provide autonomic innervations to the various urogenital organs, such as the urinary bladder, prostate, and penis. It is well established that both sympathetic and parasympathetic synaptic transmissions in autonomic ganglia are mediated mainly by acetylcholine (ACh). Until now, however, the properties of ACh-induced currents and its receptors in pelvic ganglia have not clearly been elucidated. In the present study, biophysical characteristics and molecular nature of nicotinic acetylcholine receptors (nAChRs) were studied in sympathetic and parasympathetic major pelvic ganglion (MPG) neurons. MPG neurons isolated from male rat were enzymatically dissociated, and ionic currents were recorded by using the whole cell variant patch clamp technique. Total RNA from MPG neuron was prepared, and RT-PCR analysis was performed with specific primers for subunits of nAChRs. ACh dose-dependently elicited fast inward currents in both sympathetic and parasympathetic MPG neurons (EC50; 41.4μM and 64.0μM, respectively). ACh-induced currents showed a strong inward rectification with a reversal potential near 0 mV in current-voltage relationship. Pharmacologically, mecamylamine as a selective antagonist for α3β4 nAChR potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons (IC50; 0.53μM and 0.22μM, respectively). Conversely, α- bungarotoxin, α-methyllycaconitine, and dihydro-β-erythroidine, which are known as potent and sensitive blockers for α7 or α4β2 nAChRs, below micromolar concentrations showed negligible effect. RT-PCR analysis revealed that α3 and β4 subunits were predominantly expressed in MPG neurons. We suggest that MPG neurons have nAChRs containing α3 and β4 subunits, and that their activation induces fast inward currents, possibly mediating the excitatory synaptic transmission in pelvic autonomic ganglia.