The D alpha 2 gene encodes a ligand-binding subunit of nicotinic acetylcholine receptors (nAChRs) from Drosophila melanogaster. We have studied the distribution of D alpha 2 transcripts and protein by in situ hybridization and immunohistochemistry, respectively, as well as the regulation of D alpha 2 gene expression in vivo using D alpha 2 promoter fragments fused to the Escherichia coli lacZ gene. Transcripts and protein from the D alpha 2 gene were detected exclusively in the central nervous system. Both in late embryos and adults D alpha 2-like immunoreactivity is widely but not uniformly distributed in the synaptic neuropil, suggesting that the D alpha 2 protein is a subunit of a synaptic nicotinic receptor. Its distribution resembles that of ALS and ARD proteins, two other nAChR subunits of the fly. Five different D alpha 2-lacZ fusion gene constructs were introduced into the Drosophila genome by P-element-mediated gene transfer to identity functional elements of the D alpha 2 promoter. All constructs produce a basic lacZ expression pattern that is compatible with the distribution of D alpha 2 transcripts and protein. A 880 bp upstream fragment harbors the cis elements for the expression of a weak but specific basic D alpha 2 pattern. The next 350 bp further upstream significantly enhance beta-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 D alpha 2 expression in optic lobe tangential cells are located.