Duodenal, jejunal and ileal sections from sheep were used to study in vitro absorption of valine, threonine and methionine. Everted sacs were incubated for 45 minutes at 39 C under an atmosphere of 95% O₂ - 5% CO₂ in Krebs-Ringer bicarbonate buffer (mucosal and serosal fluid) containing 5 μmoles per ml of the test amino acid. The hydrolyzed tissue and mucosal and serosal fluids were analyzed for amino acid content. The duodenum absorbed smaller quantities of amino acids from the mucosal fluid than the jejunum and significantly (P<.01) less than the ileum. The release of amino acids from duodenal tissue was small for threonine and methionine and negative for valine. Accumulation of amino acids by duodenal tissue was not significantly different from the ileum, but this represented a larger part of the amount absorbed from the mucosal fluid. The duodenum appears to be of limited importance as a site of amino acid absorption. The jejunum readily absorbed valine and methionine, but absorbed only a small amount of threonine from the mucosal fluid. The release of amino acids into the serosal fluid by the jejunum was very slight in comparison to the ileum (P<.01). Tissue accumulation of amino acids was significantly less (P<.05) than in either the duodenum or ileum. While the jejunum generally absorbed large amounts of amino acids from the mucosal fluid, it did not release or accumulate amino acids in large amounts. The ileum maximized movement of all amino acids. It absorbed significantly more (P<.01) amino acids from the mucosal fluid and released significantly more (P<.01) amino acids into the serosal fluid. The accumulation of valine and threonine by ileal tissue was significantly greater (P<.05) than the jejunum. The ileum, therefore, appears to be the most active and efficient site of valine, threonine and methionine absorption in the sheep.