Refeeding reverses the muscle protein loss seen with fasting. The physiological regulators and cellular control sites responsible for this reversal are incompletely defined. Phosphorylation of phosphorylated heat-acid stabled protein (PHAS-I) frees eukaryotic initiation factor 4E (eIF4E) and stimulates protein synthesis by accelerating translation initiation. Phosphorylation of p70 S6-kinase (p70(S6k)) is thought to be involved in the regulation of the synthesis of some ribosomsal proteins and other selected proteins with polypyrimidine clusters near the transcription start site. We examined whether phosphorylation of PHAS-I and p70(S6k) was increased by feeding and determined the separate effects of insulin and amino acids on PHAS-I and p70(S6k) phosphorylation in rat skeletal muscle in vivo. Muscle was obtained from rats fed ad libitum or fasted overnight (n = 5 each). Other fasted rats were infused with insulin (3 microU x min(-1) x kg(-1), euglycemic clamp), amino acids, or the two combined. Gastrocnemius was freeze-clamped, and PHAS-I and p70(S6k) phosphorylation was measured by quantifying the several phosphorylated forms of these proteins seen on Western blots. We observed that feeding increased phosphorylation of both PHAS-I and p70(S6k) (P < 0.05). Infusion of amino acids alone reproduced the effect of feeding. Physiological hyperinsulinemia increased p70(S6K) (P < 0.05) but not PHAS-I phosphorylation (P = 0.98). Addition of insulin to amino acid infusion was no more effective than amino acids alone in promoting PHAS-I and p70(S6k) phosphorylation. We conclude that amino acid infusion alone enhances the activation of the protein synthetic pathways in vivo in rat skeletal muscle. This effect is not dependent on increases in plasma insulin and simulates the activation of protein synthesis that accompanies normal feeding.