Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-limiting enzyme in hepatic gluconeogenesis and therefore plays a central role in glucose homeostasis. The aim of this study was to analyse the nutritional regulation of PEPCK gene expression in rainbow trout (Oncorhynchus mykiss), which are known to use dietary carbohydrates poorly. A full-length hepatic PEPCK cDNA (2637 base pairs with one open reading frame putatively encoding a 635-residue protein) was cloned and found to be highly homologous to mammalian PEPCKs. The presence of a putative peptide signal specific to a mitochondrial-type PEPCK in the deduced amino acid sequence suggests that this PEPCK gene codes for a mitochondrial form, In gluconeogenic tissues such as liver, kidney and intestine, this PEPCK gene was expressed at high levels and, in the liver we found no regulation of PEPCK gene expression by dietary carbohydrates. These results suggest that the first step of the hepatic gluconeogenic pathway in rainbow trout is functional and highly active irrespective of the dietary carbohydrate supply.