We have cloned and characterized human genes (GAD1 and GAD2) encoding the two human glutamate decarboxylases, GAD67 and GAD65. The coding region of the GAD65 gene consists of 16 exons, spanning more than 79 kb of genomic DNA. Exon 1 contains the 5' untranslated region of GAD65 mRNA, and exon 16 specifies the protein's carboxy terminal and at least part of the mRNA's 3' untranslated sequence. Similarly, the coding region of the GAD67 gene consists of 16 exons, spread over more than 45 kb of genomic DNA. The GAD67 gene contains an additional exon (exon 0) that, together with part of exon 1, specifies the 5' untranslated region of GAD67 mRNA. Exon 16 specifies the entire 3' untranslated region of GAD67 mRNA. Exons 1-3 encode the most divergent region of GAD65 and GAD67. The remaining exon-intron boundaries occur at identical positions in the two cDNAs, suggesting that they derive from a common ancestral GAD gene.