Xenopus liver contains a protein inhibitor of glucokinase that, in contrast to the mammalian regulatory protein of glucokinase, is insensitive to fructose 6-phosphate and fructose 1-phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551-556]. The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the protein purified from Xenopus liver. In contrast to the rat liver regulatory protein, both recombinant and native Xenopus regulatory proteins were insensitive to fructose 6-phosphate, fructose 1-phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucokinase with greater affinity than rat glucokinase. These results allow one to conclude that the fructose-phosphate-insensitive protein of lower vertebrates is homologous to the fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein found in mammals.