By combining a Drosophila genome data base search and reverse transcriptase-PCR-based cDNA isolation, two G-protein-coupled receptors were cloned, which are the closest known invertebrate homologs of the mammalian opioid/somatostatin receptors. However, when functionally expressed in Xenopus oocytes by injection of Drosophila orphan receptor RNAs together with a coexpressed potassium channel, neither receptor was activated by known mammalian agonists. By applying a reverse pharmacological approach, the physiological ligands were isolated from peptide extracts from adult flies and larvae. Edman sequencing and mass spectrometry of the purified ligands revealed two decapentapeptides, which differ only by an N-terminal pyroglutamate/ glutamine. The peptides align to a hormone precursor sequence of the Drosophila genome data base and are almost identical to allatostatin C from Manduca sexta. Both receptors were activated by the synthetic peptides irrespective of the N-terminal modification. Sitedirected mutagenesis of a residue in transmembrane region 3 and the loop between transmembrane regions 6 and 7 affect ligand binding, as previously described for somatostatin receptors. The two receptor genes each containing three exons and transcribed in opposite directions are separated by 80 kb with no other genes predicted between. Localization of receptor transcripts identifies a role of the new transmitter system in visual information processing as well as endocrine regulation.Insect development and behavior are largely controlled by hormones and neurotransmitters often identified using a diverse array of bioassays. Besides the biogenic amines and the steroid-like hormones, insect hormones have been frequently classified as neuropeptides, which are widely distributed throughout the invertebrate kingdom (1, 2). Despite the large number of neuropeptides, the number of known cognate receptors in insects is still rather limited, with only a few examples in Drosophila that have been cloned based on homology with mammalian G-protein-coupled receptors (GPCRs) 1 (i.e. neuropeptide Y-like and tachykinin-like receptors) (3, 4). With the completion of the Drosophila genome project, a more thorough analysis of neuropeptide/receptor relations in insects is now possible. Whereas this genome data base allows the identification of peptide hormones previously isolated from other insect species as part of larger precursors (5), Drosophila GPCR-like sequences have been predicted mostly based on structural analogy of the transmembrane regions to mammalian neuropeptide receptor groups (6).Structural evidence for the existence of ligands identical or similar to their mammalian neuropeptide counterparts are lacking when searching the Drosophila genome data base. This may indicate that in insects these receptors are activated by an entirely different set of ligands. This view is supported by data reported here on the identification of two novel GPCRs from Drosophila melanogaster, termed Drostar1 and -2, which are structurally related to the mam...