Acetyl-CoA carboxylase (ACoAC) catalyses the carboxylation of acetyl-CoA into malonyl-CoA. This product plays a pivotal role in the regulation of energy metabolism since it is both a substrate for fatty acid synthesis and an inhibitor of the oxidative pathway. The present study was initiated to analyse the modulation of ACoAC activity in liver and selected extrahepatic tissues of rainbow trout (Oncorhynchus mykiss) by dietary changes as a contribution to the understanding of the nutritional control of lipid metabolism in fish. Short-term effects of food intake were studied by measuring ACoAC activity in the liver and dorsal white muscle at different time intervals after a meal. Only slight variations were observed in the muscle during the period 2–72 h after the meal. The long-term effects of an increase in dietary lipids or carbohydrates levels were examined by measuring ACoAC activity in the liver, adipose tissue, intestine, kidney, red muscle, dorsal and ventral white muscles of trout after 3 months of feeding with different diets. ACoAC activity is stimulated by a high-digestible starch diet in the abdominal adipose tissue and the white muscle. A high-lipid diet decreases ACoAC activity in the liver and the intestine, but not in other tissues. Contrary to mammals, a rapid adaptation of ACoAC activity to food supply is not effective in rainbow trout. However, a long-term nutritional control of ACoAC activity does occur in this species, but the target tissue differs with the predominant non-protein energy sources in the diet. The present results suggest the potential existence of two ACoAC isoforms with different tissue distribution as has been observed in mammals and birds.