The gene encoding the mouse precursor of pituitary adenylate cyclase-activating polypeptide (PACAP) has been cloned, and its structural organization was determined. Using the 5'-rapid amplification of cDNA ends (RACE) procedure, three types of transcription initiation produced by alternative exon usage of two untranslated alternative exons (exons 1A and 1B) were defined. The PACAP gene spans 6.6kb of genomic DNA and is composed of six exons including the alternative exons. The signal peptide, PACAP-related peptide and mature 38-amino acid PACAP (PACAP-38) are encoded within exons 2, 4 and 5, respectively. The 5'-flanking region of the PACAP gene contains several sequence motifs homologous to cAMP response element, TPA response element, and growth hormone factor-1 binding site. A dinucleotide repeat sequence is present in an intron. In addition, there are di- and tetranucleotide repeat sequences 2.4kb and 3.2kb upstream to the translation start point, respectively. The overall intron-exon organization and the production of the alternate mRNAs of the PACAP gene are markedly similar to those of the growth hormone-releasing hormone (GHRH), supporting the hypothesis that the two genes encoding GHRH or PACAP were originated from a gene duplication. Promoter analysis of the 5'-flanking region of the PACAP gene using a luciferase gene reporter system revealed that the isolated 5'-flanking region has functional promoter activity and is responsible for inducible expression.