Rainbow trout (Oncorhynchus mykiss) are known to use dietary carbohydrates poorly. One of the hypotheses to explain the poor utilisation of dietary glucose by these fish is a dysfunction in nutritional regulation of hepatic glucose metabolism. In this study, we obtained partial clones of rainbow trout cDNAs coding for a glucose transporter (Glut2), and for the enzymes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2K/F-2,6BPase), fructose-1,6-bisphosphatase (FBPase) and pyruvate kinase (PK). Their deduced amino acid sequences were highly similar to those of mammals (up to 80% similarity). In a study of nutritional regulation, the Glut2 gene was highly expressed in the liver irrespective of the nutritional status of the trout, in agreement with the role of this transporter in the input (during refeeding) and output (during fasting) of glucose from the liver. Moreover, whereas PK and FBPase gene expression was high irrespective of the nutritional status, levels of hepatic 6PF-2K/F-2,6BPase mRNA were higher in fish fed with carbohydrates than in fish deprived of food. The high levels of hepatic PK, Glut2 and 6PF-2K/F-2,6BPase gene expression observed in this study suggest a high potential for tissue carbohydrate utilisation in rainbow trout. The persistence of a high level of FBPase gene expression suggests an absence of regulation of the gluconeogenic pathway by dietary carbohydrates.