The effects of insulin and IGF-I on fatty acid (FA) and glucose metabolism were examined using oleic acid or glucose as tracers in differentiated rainbow trout ( Oncorhynchus mykiss ) myotubes. Insulin and IGF-I significantly reduced the production of CO₂from oleic acid with respect to the control values. IGF-I also significantly reduced the production of acid-soluble products (ASP) and the concentration of FA in the medium, while cellular triacylglycerols (TAG) tended to increase. Only insulin produced a significant accumulation of glycogen inside the cells in glucose distribution experiments. Incubation with catecholamines did not affect oleic acid metabolism. Cells treated with rapamycin [a target of rapamycin (TOR) inhibitor] significantly increased the oxidation of oleic acid to CO₂and ASP, while the accumulation of TAG diminished. Rosiglitazone (a peroxisome proliferator-activated receptor γ agonist) and etomoxir (a CPT-1 inhibitor) produced a severe and significant reduction in the production of CO₂and ASP. Rosiglitazone and etomoxir also produced a significant accumulation of FA outside and inside the cells, respectively. No significant effects of these drugs on glucose distribution were observed. These data indicate that insulin and IGF-I act as anabolic hormones in trout myotubes in both oleic acid and glucose metabolism, although glucose oxidation appears to be less sensitive than FA oxidation to insulin and IGF-I. The use of rapamycin, etomoxir, and rosiglitazone may help us to understand the mechanisms of regulation of lipid metabolism in fish.