Intestinal digestion of two tripeptides (leucyl-glycyl-glycine, prolyl-glycyl-glycine) and two disacchrarides (sucrose, maltose) was examined in the hamster by intestinal perfusion in vivo and hydrolysis of the substrates by microvillus membranes. Perfusion studies showed that luminal disappearance rates of leucyl-glycl-glycine were significantly higher than prolyl-glycyl-glycine (P less than o.001), sucrose (P less than 0.001), and maltose (P less than 0.005). Hydrolytic products of leucyl-glycyl-glycine, sucrose, and maltose were detected in the gut lumen in appreciable concentrations, whereas negligible concentrations of prolyl-glycyl-glycine products were present. Leucyl-glycyl-glycine hydrolysis in microvillus membranes was markedly higher than prolyl-glycyl-glycine (P less than 0.001), which was predominant in the cytoplasmic fraction. These results indicate that leucyl-glycyl-glycine, like sucrose and maltose, is hydrolyzed at the membrane. With some tripeptides, i.e., leucyl-glycyl-glycine, digestion occurs at the microvillus membrane with subsequent transport of hydrolytic products into the intestinal epithelial cell. Other tripeptides, i.e., prolyl-glycyl-glycine, may cross the membrane and undergo intracellular hydrolysis by cytoplasmic peptidases.