An extract of adult Drosophila melanogaster was separated by gel exclusion, ion exchange, and reversed-phase chromatography. Four peptides, each with an -ArgPheNH2 C-terminal sequence, were identified by radioimmunoassay. The primary sequences were determined by Edman degradation and confirmed by mass spectrometry and sequence-specific radioimmunoassay. Three of the peptides are encoded by Drosophila proFMRFamide: AspProLysGlnAspPheMetArgPheNH2 (DPKQDFMRFamide), ThrProAlaGluAspPheMetArgPheNH2 (TPAEDFMRFamide), and SerAspAsnPheMetArgPheNH2 (SDNFMRFamide). A novel Drosophila peptide ThrAspValAspHisValPheLeuArgPheNH2 (TDVDHVFLRFamide) was also isolated. TDVDHVFLRFamide is structurally related to peptides isolated from chicken, cockroach, locust, and snail; the cockroach, fruitfly, and locust peptides differ only by the N-terminal amino acid residue. Two Drosophila neural genes, dsk and FMRFamide, are known to encode -ArgPheNH2-containing peptides; however, neither encodes TDVDHVFLRFamide, indicating that Drosophila contains another precursor encoding -ArgPheNH2 peptides.