The Est-6 gene of Drosophila melanogaster was cloned by screening libraries with synthetic oligonucleotides corresponding to tryptic peptides from purified esterase-6 (Est-6) protein. cDNA clones were isolated that hybridized in situ to the site of Est-6 on chromosome 3 at 69A1. Inserts in putative Est-6 cDNA clones were 1.85 kilobases (kb) long, and blot hybridization analysis of electrophoretically fractionated RNA, using a cDNA clone as a probe, revealed two transcripts, of 1.68 and 1.83 kb. The two transcripts showed the same developmental profile as the Est-6 protein. Neither transcript was detected in an Est-6-null line. The cDNA fragment was homologous to a 2.3-kb EcoRI-BamHI fragment in genomic clones, and this region was interrupted by the 8-kb B104 transposable element in the Est-6-null line. Conceptual translation of the cDNA sequence revealed a protein of 548 residues with 19% sequence similarity to acetylcholinesterase from the Torpedo ray.