The intestinal transport of the dipeptide prolylhydroxyproline was investigated using hamsters and segments of hamster small intestines. The feeding of prolylhydroxyproline to hamsters resulted in the urinary excretion of 2% of the ingested dipeptide after 3 h or 7% after 24 h. In vitro perfusions of prolylhydroxyproline through the hamster small intestine resulted in the movement of the dipeptide across the intestinal wall. The rate of absorption was proportional to the concentration of prolylhydroxyproline on the mucosal side (0.17 μmol Pro∙Hyp/mM in lumen/g tissue/h). Anoxia, 2,4-dinitrophenol, cyanide, or an excess of proline or hydroxyproline did not change this absorption rate. These results suggest that prolylhydroxyproline is absorbed by "simple passive diffusion", that it reaches the site of intestinal absorption because of its relative resistance to enzymatic hydrolysis, and that it is excreted by glomerular filtration without subsequent active reabsorption.