In the enteric nervous system (ENS) excitatory nicotinic cholinergic transmission is mediated by neuronal nicotinic acetylcholine receptors (nAChR) and is critical for the regulation of gastric motility. nAChRs are ligand-gated pentameric ion channels found in the CNS and peripheral nervous system. The expression of heteromeric nAChR and receptor subunit mRNAs was investigated in the neonatal rat ENS using receptor autoradiography with the radiolabeled ligand (125)I-epibatidine, and in situ hybridization with subtype specific probes for ligand binding alpha (alpha2, alpha3, alpha4, alpha5, alpha6) and structural beta (beta2, beta3, beta4) subunits. The results showed strong nicotine sensitive binding of (125)I-epibatidine around the stomach, and small and large intestines. The binding was partially displaced by A85380, a nicotinic ligand which differentiates between different heteromeric nAChR subtypes, suggesting a mixed receptor population. Radioactive in situ hybridization detected expression of alpha3, alpha5, alpha7, beta2 and beta4 mRNA in the myenteric plexus of the stomach, and small and large intestines. In the submucosal plexus of the small and large intestines expression of alpha3, alpha5 and beta4 was found in some ganglia. There was no signal for alpha4, alpha6 and beta3 in the ENS but positive hybridization signal for alpha2 transcripts was seen in some areas of the small intestines. However, the signal was not associated with any ganglion cells. The results confirm the presence of heteromeric nAChRs in the ENS similar to those found in the peripheral nervous system, with the majority being composed of alpha3(alpha5)beta4, and a few alpha3beta2 nAChRs. In addition, homomeric alpha7 nAChRs could be present.