The P1 gene codes for a major RNA, which accumulates specifically in the fat body cells at the late third larval stage of Drosophila melanogaster development under the positive control of the insect molting hormone 20-hydroxyecdysone. The primary structure of the P1 gene and the 5' upstream flanking region to position -776 relative to the transcription start was determined by sequence analysis of a cloned genomic DNA segment and two cDNAs containing sequences complementary to the 5' and 3' ends of the P1 transcript. The RNA coding region spans 3469 nucleotides and contains a 59-base-pair intron close to its 5' end, as predicted by computer analysis and established by S1 nuclease protection, primer extension and cDNA sequencing. The predicted P1 polypeptide contains 1030 amino acids, including a putative 16-amino acid signal peptide and two stretches of 12 and 11 aspartic and asparagine residues. Short stretches of nucleotide sequences similar to sequences located in the 5' regions of other genes expressed in the D. melanogaster fat body were found in the proximal promoter and transcribed region of the P1 gene.