Gastrin-releasing peptide (GRP), the mammalian homolog of the amphibian peptide bombesin, is encoded in man by a single gene located on chromosome 18. Restriction enzyme and DNA sequence analyses establish that the gene is 10 kilobases in size with two introns of 4.8 and 3.9 kilobases. Exon 1 encodes the 5'-untranslated region, the signal peptide, and the first 23 amino acids of GRP. Exon 2 encodes the remaining three complete amino acids of GRP and the first 74 amino acids of the GRP carboxy-terminal extension peptide. Hence, intron 1 interrupts the coding region of the bioactive portion of GRP between the first and second nucleotides for Gly, the 24th amino acid of GRP. Exon 3 encodes the remainder of the GRP-extension peptide and the 3'-untranslated region. Two GC-rich, potential regulatory sequences and a sequence associated with regulation by cAMP lie between the CAAT and TATA boxes; the primary transcriptional start site is located 30 bases downstream from the TATA box. The second intron has an alternate donor site at its 5'-end and an alternate acceptor site at its 3'-end. S1 nuclease mapping demonstrates that differential RNA splicing using these sites results in the similar expression of three GRP mRNAs in GRP-containing neurons (in stomach and brain) as well as in GRP-containing neuroendocrine cells (fetal lung). In addition, the pattern of RNA splicing is similar between normal tissue and neoplastic tissue (small cell carcinoma of the lung and medullary carcinoma of the thyroid).