The insect allatostatins are neurohormones, acting on the corpora allata (where they block the release of juvenile hormone) and on the insect gut (where they block smooth muscle contraction). We screened the "Drosophila Genome Project" database with electronic sequences corresponding to various insect allatostatins. This resulted in alignment with a DNA sequence coding for some Drosophila allatostatins (drostatins). Using PCR with oligonucleotide primers directed against the presumed exons of this Drosophila allatostatin gene and subsequent 3'- and 5'-RACE, we were able to clone its cDNA. The Drosophila allatostatin preprohormone contains four amino acid sequences that after processing would give rise to four Drosophila allatostatins: Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH(2) (drostatin-1), Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH(2) (drostatin-2), Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH(2) (drostatin-3), and Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH(2) (drostatin-4). Drostatin-2 is identical to helicostatin-2 (11-18) and drostatin-3 to helicostatin-3, two neurohormones previously isolated from the moth Helicoverpa armigera. Furthermore, drostatin-3 has previously been isolated from Drosophila itself. Drostatins-1 and -4 are novel members of the insect allatostatin neuropeptide family. The Drosophila allatostatin preprohormone gene contains two introns and three exons. The gene is located on the right arm of the third chromosome, position 96A-B. The existence of at least four different Drosophila allatostatins opens the possibility of a differential action of some of these hormones on the two recently cloned Drosophila allatostatin receptors, DAR-1 and -2. This is the first report on an allatostatin preprohormone from Drosophila.