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Plasma glucose and immunoreactive insulin (IRI) concentrations and activities of enzymes related to glucose metabolism in livers were measured in dogs and cats. Nucleotide sequences of the conserved region of glucokinase (GK) cDNA that contained ATP- and glucose-binding domains were determined in canine liver and feline pancreas for design of the species-specific oligonucleotide primers for reverse transcription–polymerase chain reaction (RT-PCR) analysis. There were no significant differences in plasma glucose and IRI concentrations between dogs and cats. In feline liver, although GK activities were not detected, activities of hexokinase, fructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, fructose-1,6-bisphosphatase and glucose-6-phosphatase were significantly higher than those in canine liver. The partial sequences of canine liver GK and feline pancreas GK cDNA were respectively 88% and 89% identical with the rat liver GK cDNA. Expression of GK gene was observed in canine liver and pancreas and feline pancreas with RT-PCR using species specific primers based on the cDNA sequences.