AbstractHeteropentameric nicotinic acetylcholine receptors (nAChR) mediate fast synaptic transmission in ganglia of the autonomic nervous system. It is undisputed that α3 and β4 are the predominant subunits in the superior cervical ganglion (SCG); however, reports on the presence of receptors that contain α4 have been controversial. Here, we have searched for the presence of α4‐containing nAChRs in the postnatal rat and mouse SCG. We now show by immunoprecipitation combined with radioligand binding that α4‐containing receptors constitute about 20% of hetero‐oligomeric nAChRs in postnatal Day 3 (P3) mice. However, already by P9, the level of α4 approaches zero. In contrast, the number of α4‐containing receptors is close to zero in the rat SCG at all times investigated. Deletion of the β2 subunit by using α5β2‐double knockout (KO) mice removes all α4‐containing receptors, suggesting that in the postnatal mouse SCG, α4 co‐assembles only with β2 but not with β4. α4β2 receptors are, on the other hand, up‐regulated in the SCG of P3 α5β4‐double KO mice, where they make up about 50% of receptors that bind [3H]‐epibatidine. Nonetheless, receptors on the surface of SCG neurons from α5β4‐double KO mice maintained for one to two days in culture comprise <10% of α4β2 and >90% of α3β2, as determined by patch clamp recordings with α4β2‐ and α3β2‐specific ligands. We propose that in the P3 SCG of wild type mice, α3β4 (±α5) represent about 62% of receptors, whereas 17% are α3β2β4, and 21% are α4β2 (±α5) receptors. © 2011 Wiley Periodicals, Inc. Develop Neurobiol 71: 390–399, 2011