The expression patterns of three clustered neuronal nicotinic acetylcholine receptor (nAchR) subunit genes ordered β4, α3, and α5 overlap extensively in the peripheral nervous system (PNS) but only partially in the central nervous system (CNS). We have begun to investigate cell type-specific cis elements regulating these genes by analyzing in both cell culture and transgenic mice, a 2.8-kb fragment (−2732/+47) containing the α3 promoter region, the β4/α3 intergenic region, and a portion of the β4 3′-untranslated exon. The −2732/+47 fragment is preferentially active in PC12 cells relative to nonneural cell lines. Deletion analysis revealed a cell type-specific positive transcriptional element positioned in the β4 3′-untranslated exon. The positive element is likely to be an enhancer and not a second α3 promoter, because no α3 exons are present in this region. Having shown in cell culture that cell-type specific cis elements are positioned between the β4 and α3 coding regions, we investigated the activity of −2732/+47 in vivo. Transgenic mice were generated, which carry the lacZ gene fused downstream of −2732/+47. Expression of the lacZ transgene is restricted to neurons of the CNS; no expression was detected in the PNS or in nonneural tissues. LacZ-positive cells were detected virtually exclusively in a subset of CNS nuclei that transcribe the endogenous α3 gene. Some overlap was seen with the β4 gene, but nearly none with the α5 gene. Our results demonstrate that cis elements positioned between the α3 and β4 coding regions are important for establishing part of the restricted CNS patterns of β4, α3, and α5 gene transcription. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 311–324, 1997