The Dα2 gene encodes a ligand-binding subunit of nicotinic acetylcholine receptors (nAChRs) from Drosophila melanogaster. We have studied the distribution of Dα2 transcripts and protein by in situ hybridization and immunohistochemistry, respectively, as well as the regulation of Dα2 gene expression in vivo using Dα2 promoter fragments fused to the Escherichia coli lacZ gene. transcripts and protein from the Dα2 gene were detected exclusively in the central nervous system. Both in late embryos and adults Dα2-like immunoreactivity is widely but not uniformly distributed in the synaptic neuropil, suggesting that the Dα2protein is a subunit of a synaptic nicotinic receptor. Its. distribution resembles that of ALS and ARD protein, two other nAChR subunits of the fly. Five different Dα2-lacZ fusion gene constructs were introduced into theDrosophila genome by P-element–mediated gene transfer to identity functional elements of the Dα2 promoter. All constructs produce a basic lacZ expression pattern that is compatible with the distribution of Dα2 transcripts and protein. A 880 bp upstream fragment harbors the cis elements for the expression of a weak but specific basic Dα2 pattern. The next 350 bp further upstream significantly enhance β-galactosidase expression without influencing the pattern of expression. Between 1.7 and 7.3 kb upstream of the transcription start site one or more elements that are required for Dα2 expression in optic lobe tangential cells are located. 1994 John Wiley & Sons, Inc.