In vivo studies have reported conflicting effects of insulin on mixed tissue protein synthesis rates. To test the hypothesis that insulin has differential effects on synthesis rates of various protein fractions in different organs, we infused miniature swine (n = 8 per group) with saline, insulin alone (at 0.7 mU/kg(-1). min(-1)), or insulin plus an amino acid mixture for 8 h. Fractional synthesis rate (FSR) of mitochondrial and cytoplasmic proteins in liver, heart, and skeletal muscle, as well as myosin heavy chain (MHC) in muscle, were measured using L-[1-(13)C]leucine as a tracer. The FSR of mitochondrial and cytoplasmic proteins were highest in liver, followed by heart and then muscle. Mitochondrial FSR in muscle was higher during insulin and insulin plus amino acid infusions than during saline. Insulin had no significant effect on FSR of MHC in muscle. In contrast, FSR of both mitochondrial and cytoplasmic proteins were not stimulated by insulin in liver. Insulin also did not increase FSR of mitochondrial in heart, whereas insulin and amino acid stimulated FSR of cytoplasmic protein. In conclusion, insulin stimulates the synthesis of muscle mitochondrial proteins, with no significant stimulatory effect on synthesis of sarcoplasmic and MHC. These results demonstrate that insulin has different effects on synthesis rates of specific protein fractions in the liver, heart, and skeletal muscle.