The potential roles of insulin and dietary amino acids in the regulation of skeletal muscle protein synthesis were examined in adult and old rats. Animals were fed over I h with either a 25% or a 0% amino acid/protein meal. In each nutritional condition, postprandial insulin secretion was either maintained or blocked with diazoxide injections. Protein synthesis in gastrocnemius and soleus muscles was assessed in vivo using the flooding dose method. Insulin suppression decreased protein synthesis in both muscles irrespective of the nutritional condition and age of the rats. Moreover, reduced insulinaemia was associated with 4E-BP1 dephosphorylation, enhanced assembly of the 4E-BP1 -eIF4E inactive complex and hypophosphorylation of eIF4E, p70(S6k) and protein kinase B, key intermediates in the regulation of translation initiation and protein synthesis. Old rats did not differ from adult rats. The lack of amino acids in the meal of insulin-suppressed rats did not result in any additional decrease in protein synthesis. In the presence of insulin secretion, dietary amino acid suppression significantly decreased gastrocnemius protein synthesis in adult but not in old rats. Amino acid suppression was associated with reduced phosphorylation of 4E-BPI and p70(S6k) in adults. Along with protein synthesis, only the inhibition of p70(S6k) phosphorylation was abolished in old rats. We concluded that insulin is required for the regulation of muscle protein synthesis irrespective of age and that the effect of dietary amino acids is blunted in old rats.