Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in muscle and liver of pigs parenterally infused with amino acids and insulin. To examine the effects of enteral protein and carbohydrate on protein synthesis, pigs (n = 42, 1.7 kg body wt) were fed isocaloric milk diets containing three levels of protein (5, 15, and 25 g x kg body wt(-1) x day(-1)) and two levels of lactose (low = 11 and high = 23 g x kg body wt(-1) x day(-1)) from 1 to 6 days of age. On day 7, pigs were gavage fed after 4-h food deprivation, and tissue protein synthesis rates and biomarkers of mRNA translation were assessed. Piglet growth and protein synthesis rates in muscle and liver increased with dietary protein and plateaued at 15 g x kg body wt(-1) x day(-1) (P < 0.001). Growth tended to be greater in high-lactose-fed pigs (P = 0.07). Plasma insulin was lowest in pigs fed 5 g x kg body wt(-1) x day(-1) protein (P < 0.0001). Plasma branched-chain amino acids increased as protein intake increased (P < 0.0001). Muscle (P < 0.001) and liver (P < or = 0.001) ribosomal protein S6 kinase-1 and eIF4E-binding protein phosphorylation increased with protein intake and plateaued at 15 g x kg body wt(-1) x day(-1). The results indicate that growth and protein synthesis rates in neonatal pigs are influenced by dietary protein and lactose intake and might be mediated by plasma amino acids and insulin levels. However, feeding protein well above the piglet's requirement does not further stimulate the activation of translation initiation or protein synthesis in skeletal muscle and liver.