Two different preparations of vascularly-perfused rat small intestine are described and their application to metabolic studies in this tissue illustrated. The first, a totally isolated, blood-perfused intestinal preparation, has an apparent requirement for glucocorticoid and norepinephrine. Lymph is produced and collected, permitting studies of fat transport and lipoprotein biosynthesis. Results indicate that intestine synthesizes some, but not all, of the proteins associated with chylomicrons and other lipoproteins of intestinal lymph. The isolated intestine from 260-g rats metabolizes 75 mumoles of circulating glutamine per hour, mostly to CO2, lactate, citrulline, proline, alanine, and ammonia. In a second preparation, all venous blood is collected from a short, isolated intestinal segment in situ with intact arterial blood supply. Adaptations allow the quantitative determination of rates of uptake and metabolism of substances from both blood and lumen. Results indicate that over 36% of CO2 produced by intestine of fasted rats is derived from plasma glutamine and only 8% from glucose. Virtually all glutamate and aspartate and significant amounts of glutamine and arginine absorbed from the lumen are also metabolized. The resulting metabolic products were quantitated and include CO2, lactate, proline, citrulline, alanine, and glucose.