cDNA clones coding for the catalytic subunit of rat liver glucose-6-phosphatase (EC 3.1.3.9) were isolated from a rat liver cDNA library in lambda gt11 phage. The sequence of the cDNA and the amino acid sequence derived from it were greater than 90% identical to the corresponding sequences for the mouse and human forms of liver glucose-6-phosphatase. Northern blot analysis of RNA from FAO hepatoma cells revealed that dexamethasone induced the glucose-6-phosphatase mRNA while insulin suppressed its expression. When both hormones were added together insulin completely suppressed the effect of glucocorticoid. cAMP addition alone decreased the abundance of glucose-6-phosphatase mRNA. The results demonstrate multihormonal regulation of gene expression of hepatic glucose-6-phosphatase and support a dominant role for insulin.