We have cloned and determined the complete structure of the murine gene encoding the 67-kD form of glutamic acid decarboxylase (GAD67), the gamma-aminobutyric acid synthetic enzyme. Its coding region comprises 18 exons spanning 42 kb of genomic DNA. Exon 1 together with 64 bp of exon 2 defines the 5' untranslated region of GAD67 mRNA. Exon 18 specifies the protein's carboxyl terminal and the entire 3' untranslated region. Exons 7/A and 7/B are solely contained in the coding regions of two alternatively spliced bicistronic embryonic mRNAs, which code for the truncated embryonic GAD forms. The promoter region (P1) corresponding to the main group of transcription initiation sites is devoid of TATA and CAAT boxes but has putative binding sites for the transcription factor SP1 and is embedded in a large G + C-rich domain of a CpG island, features shared by the promoters of constitutively expressed housekeeping genes. Primer extension data suggests the existence of additional transcription start sites at 130 bp and 295 bp upstream from the major initiation site that are utilized less frequently in adult brain. The tentative distal promoters (P2 and P3) that correspond to the minor start sites resemble tissue-specific promoters with TATA and CAAT-like boxes. In 1.3 kb of the 5'-upstream region, we identified several putative transcription factor binding sites such as AP2, Hox, E-box, egr-1, and NF-kappaB and putative neuronal-specific regulatory elements, including the neuronal-restrictive silencer element, which may have functional significance in the developmental and tissue-specific expression of the GAD67 gene.