The gene encoding rat fructose-1,6-bisphosphatase was isolated from a rat genomic Charon 4A library by screening with a cDNA to the rat liver enzyme. Southern blotting of rat genomic DNA showed that there is a single copy of the fructose-1,6-bisphosphatase gene. It extends over 23 kilobases and is composed of seven exons and six introns that range in size from 93 to 267 base pairs and from 1,400 to 11,300 base pairs, respectively. The intron/exon boundary sequences conform to consensus acceptor (GTn) and donor (nAG) sequences, and the exons in the gene appear to code for functional protein domains. The transcription start site, determined by 5'-extension sequencing of mRNA, was assigned to a guanine 119 bases 5' to the translation initiation AUG. The sequence of the gene upstream to the cap site contains characteristic RNA polymerase II promoter-binding sites: a putative TATA box at position -29 and a Sp 1 binding site (GGGGCGGAGA) at position -48. A 1,300-base pair fragment of 5'-flanking sequence containing these elements, ligated upstream from a firefly luciferase reporter gene and transfected into cultured normal rat kidney cells, demonstrated strong promoter activity. The accumulation of fructose-1,6-bisphosphatase mRNA in hepatocytes incubated with cAMP suggests that the gene may be cAMP-responsive, which is consistent with the presence of three consensus cAMP regulatory elements at positions -169, -282, and -698 in the 5'-flanking region of the gene. Expression of the fructose-1,6-bisphosphatase promoter-driven luciferaes gene was 2-3-fold activated by the cyclic nucleotide, suggesting that one or more of these elements may be functional. On the other hand, insulin decreased the expression of the endogenous gene in hepatocytes. Thus, expression of the fructose-1,6-bisphosphatase gene is regulated independently by both cAMP and insulin.