d-Fructose-1,6-bisphosphate 1-phosphohydrolase [EC 3.1.3.11] (Fru-1,6P 2ase), a regulatory enzyme of gluconeogenesis, was isolated from Rana esculenta liver in homogeneous form with approximately 30% yield. Basic kinetic properties of the enzyme and its subunit molecular weight were determined. K m is 1.72 μM. Like other vertebrate Fru-1,6P 2ase, the frog liver enzyme is inhibited by fructose-2,6-bisphosphate (Fru-2,6P 2) competitively, K i is 78 nM and by AMP allosterically, I 0.5 is 10.9 μM. Both inhibitors (Fru-2,6P 2 and AMP) act synergistically on liver Fru-1,6-P 2ase. K i for Fru-2,6P 2 determined in the presence of 1–10 μM of AMP were 35–2 nM, respectively. Maximum activity was found at pH 7.5. Like other Fru-1,6P 2ases, the frog enzyme requires magnesium ions for its activity and is activated by potassium ions; the K a for Mg 2+ is 267 μM, K a for K + is 77 mM. The subunit molecular weight of the frog liver Fru-1,6P 2ase was 37 300 Da. A great similarity between regulatory properties of frog liver Fru-1,6P 2ase and liver enzymes of other vertebrates, suggests a similar regulation of gluconeogenesis in amphibia and other vertebrates.