The Ace locus of the Drosophila genome controls biosynthesis of the neurotransmitter-hydrolyzing enzyme acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). We injected the mRNA species hybridizing with DNA fragments from this region into Xenopus oocytes, in which acetylcholinesterase mRNA is translated into active acetylcholinesterase. A 2.0-kilobase (kb) fragment of DNA from this region selectively hybridizes with Drosophila mRNA capable of inducing the biosynthesis of acetylcholinesterase in oocytes. This Drosophila DNA fragment cross-hybridized with human brain poly(A)+ RNA. We therefore used this DNA fragment as a probe for homologous sequence(s) in a human genomic DNA library and thus selected a 13.5-kb human DNA segment. DNA blot-hybridization revealed that a 2.6-kb fragment of this human DNA segment hybridizes with the Drosophila 2.0-kb DNA fragment. Both Drosophila and human fragments hybridized with a human brain mRNA species of about 7.0-kb that was barely detectable in the acetylcholinesterase-deficient HEp carcinoma. A fraction containing mRNA of similar size, extracted from human brain, induced acetylcholinesterase biosynthesis in oocytes. The human DNA fragment also was used in hybridization-selection experiments. In oocytes, hybrid-selected human brain mRNA induced acetylcholinesterase activity that was completely inhibited by 1,5-bis[4-allyldimethylammonium)phenyl]pentan-3-one dibromide but not by tetraisopropyl pyrophosphamide, a differential response to these inhibitors characteristic of "true" human brain acetylcholinesterase. These findings strongly suggest that both the Drosophila and the human DNA fragments are directly involved in controlling acetylcholinesterase biosynthesis.