To determine the effect of parenteral nutrition on the balance and catabolism of leucine (by oxidation) and phenylalanine (by hydroxylation) and to assess any acute changes in proteolysis and/or protein synthesis, leucine and phenylalanine kinetics were measured by stable isotope tracer infusions in nine 32-wk gestation premature infants under both basal conditions and in response to an i.v. infusion of glucose, lipid, and amino acids. Leucine and phenylalanine balance both changed from negative to positive during parenteral nutrition. However, leucine and phenylalanine catabolism were differently affected by parenteral nutrition; the rate of leucine oxidation increased 2-fold, whereas the rate of phenylalanine hydroxylation was unchanged from basal values. Phenylalanine utilization for protein synthesis and leucine utilization for protein synthesis (based on both plasma leucine and alpha-ketoisocaproic acid enrichments) increased significantly during parenteral nutrition. The endogenous rates of release of leucine (based on plasma leucine enrichment) and phenylalanine (both reflecting proteolysis) were significantly reduced during parenteral nutrition. The endogenous rate of release of leucine (based on alpha-ketoisocaproic acid enrichment) was slightly but not significantly lower during parenteral nutrition. The substantial increase in leucine oxidation without changes in phenylalanine hydroxylation suggests a possible limitation in the phenylalanine/tyrosine supply during parenteral nutrition. In addition, these results suggest that premature infants respond to parenteral nutrition with acute increases in whole body protein synthesis as well as a probable reduction in proteolysis.