Acetylcholinesterase is a key component of cholinergic neurotransmission. In Drosophila melanogaster, acetylcholinesterase is encoded by the Ace locus. We have determined the complete organization of the locus. The transcription unit is 34 kb (1 kb = 10(3) bases) long and encompasses ten exons. We have mapped the 5' end of the transcript, sequenced all the intron/exon boundaries, as well as the 3' end of the transcript. The deduced mature transcript is 4291 nucleotides long without poly(A). Sequencing of the promoter region reveals a potential TATA box and (GA)n motives. The Drosophila coding sequence is more split than its vertebrate counterparts, but the splicing sites of the two last exons are precisely conserved among Drosophila and vertebrate cholinesterases, and intriguingly also with the bovine thyroglobulin gene. Finally, a number of the mutations isolated in earlier genetic work are precisely placed on our molecular map in introns, exons and promoter regions. Among them, for example, a short deletion known to affect acetylcholinesterase level and tissue distribution removes promoter regions and the first non-coding exon.