The gene for an NAD-specific glutamate dehydrogenase (NAD-GDH) that is allosterically activated by NADP+ (non-substrate) was cloned, and its physical structure and nucleotide sequence was determined. The gene consists of 9 introns and 10 exons; the 10th and largest exon, which is 1863 nucleotides long, is at the 3'-end of the gene. The shortest exon of 33 base pairs is the first and is located at the 5'-end of the gene. The large exon is in perfect register along the complementary strand with a heat shock 70 (HSP)-like protein gene. The NAD-GDH gene is inducible with L-glutamine, just as the HSP 70-like protein gene (LéJohn, H.B., Cameron, L.E., Yang, B., MacBeath, G., Barker, D.S., and Williams, S.A. (1994) J. Biol. Chem. 269, 4513-4522). The phenomenon of anti-parallel coupling of two genes is named antisense gene pair. By Northern and Western blotting techniques, we obtained indirect evidence that the gene is expressed in vivo. The gene encodes a protein of M(r) 118,740 which consists of 1063 amino acid residues. The 5' and 3' borders of the gene display typical but unproven promoter motifs of CCAAT, TATAAT, and AAATAAAA polyadenylation signal bounded by a pyrimidine-rich transcription termination-type format. Restriction endonuclease site mapping of all the genomic clones isolated that carry most or all of the gene, and of the genome itself, gave hybridization patterns that are consistent with the interpretation that the organism, Achlya klebsiana, has only one form of the gene. 3'-End-labeling of a 5.2-kb XbaI DNA fragment (carrying the antisense gene pair) that was then asymmetrically cleaved to produce two single 3'-end-labeled pieces that were used as probes on L-glutamine-induced cell poly(A)+ RNA, showed that the end-labeled DNA equivalent to the HSP 70-like protein mRNA hybridized to a 3.4-kb transcript and the end-labeled DNA equivalent to the NAD-GDH mRNA hybridized to a 2.4-kb transcript.