The metabolism of endogenous and exogenous amino acids has been characterized during a 16-h fast after birth in the rat. Eighteen of 22 amino acids showed a decrease in plasma concentration up to 16 h, the most profound and sustained changes affecting those quantitatively important in gluconeogenesis. The hepatic accumulation of injected [14C]aminoisobutyric acid showed a progressive rise after birth. The in vivo conversion of 14C-labeled lactate, alanine, serine, and glutamine to [14C]glucose increased for 6 h, but all except glutamine showed a decline by 16 h. The in vitro conversion of several gluconeogenic substrates (10mM), however, increased with time in each instance. These data confirm that the capacity for hepatic gluconeogenesis and maintenance of blood glucose concentration appears immediately after birth. Nevertheless, profound hypoglycemia recurs at 16 h and responds only minimally and transiently to exogenous gluconeogenic substrate loads. In contrast, the fed newborn maintains normoglycemia, higher endogenous amino acid levels, and the capacity for substrate conversion at this time. The mechanism for stimulation of hepatic gluconeogenic pathways thus is present in both fasted and fed neonatal rats. However, owing to insufficient energy sources to sustain gluconeogenesis and to inadequate gluconeogenic substrate, the rat is unable to maintain normoglycemia if fasted 16 h.