G-protein modulation of neuronal nicotinic acetylcholine receptor (nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTPgammaS increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh- and nicotine-evoked current amplitudes were increased approximately twofold in the presence of GTPgammaS. In inside-out membrane patches, the open probability (NP(o)) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2 mm GTPgammaS relative to control but was unchanged in the presence of GDPbetaS. The modulation of nAChR-mediated whole-cell currents was agonist specific; currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTPgammaS. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G(o)alpha or Gbetagamma subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nm Gbetagamma increased the open probability of ACh-activated single-channel currents fivefold, whereas G(o)alpha (50 nm) produced no significant increase in NP(o). Neuronal nAChR subunits alpha3-alpha5 and beta2 exhibited a positive interaction with G(o)alpha and Gbetagamma, whereas beta4 and alpha7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.