Hypocitrullinemia and hypoargininemia but hyperprolinemia are associated with elevated plasma concentration of lactate in infants. Because the small intestine may be a major organ for initiating proline catabolism via proline oxidase in the body and is the major source of circulating citrulline and arginine in neonates, we hypothesized that lactate is an inhibitor of intestinal synthesis of citrulline and arginine from proline. To test this hypothesis, jejunum was obtained from 14-day-old suckling pigs for preparation of enterocyte mitochondria and metabolic studies. Mitochondria were used for measuring proline oxidase activity in the presence of 0–10 mMl-lactate. For metabolic studies, enterocytes were incubated at 37°C for 30 min in Krebs bicarbonate buffer (pH 7.4) containing 5 mMd-glucose, 2 mMl-glutamine, 2 mMl-[U-14C]proline, and 0, 1, 5, or 10 mM l-lactate. Kinetics analysis revealed noncompetitive inhibition of intestinal proline oxidase by lactate (decreased maximal velocity and unaltered Michaelis constant). Lactate had no effect on either activities of other enzymes for arginine synthesis from proline or proline uptake by enterocytes but decreased the synthesis of ornithine, citrulline, and arginine from proline in a concentration-dependent manner. These results demonstrate that lactate decreased intestinal synthesis of citrulline and arginine from proline via an inhibition of proline oxidase and provide a biochemical basis for explaining hyperprolinemia, hypocitrullinemia, and hypoargininemia in infants with hyperlactacidemia.