The effects of replacing a digestible energy source from fat (fish oil) with carbohydrate (wheat starch) on performance, glycogenesis and de novo lipogenesis was examined in triplicate groups of juvenile gilthead sea bream (Sparus aurata), fed four extruded experimental diets. In order to trace the metabolic fate of dietary starch, 0.7% wheat starch was replaced with isotope-labelled starch (>98% 13C). Fish were fed the experimental diets for three consecutive 10 d periods, and isotope ratio MS was applied to quantify 13C enrichment of liver and whole-body glycogen and lipid pools over the three feeding periods. Glycogenesis originating from dietary starch accounted for up to 68.8 and 38.8% of the liver and whole-body glycogen pools, respectively, while up to 16.7% of the liver lipid could be attributed to dietary starch. Between 5 and 8% of dietary starch carbon was recovered in whole-body lipid, and estimated deposition rates of de novo synthesised lipid originating from starch ranged from 18.7 to 123.7 mg/kg biomass per d. Dietary treatments did not significantly affect growth, feed performance or body composition of the fish, while the hepatosomatic index and glycogen content of whole fish and livers correlated directly with dietary starch inclusion level. The study suggests that gilthead sea bream efficiently synthesises glycogen from both dietary starch and endogenous sources. In contrast, lipogenesis from carbon derived from starch seems to play a minor role in overall lipid synthesis and deposition under the specified experimental conditions.