Using a freezing point depression method osmolality in the intestinal tissue of four mammals (gerbils, guinea-pigs, rabbits and rats) was estimated in vivo, during fluid transport from an isotonic electrolyte-glucose solution. Net fluid transport was also measured. In gerbils, guinea-pigs and rabbits tissue osmolality was also estimated during in vitro conditions. A marked hyperosmolality was observed in vivo in the upper parts of the villi of all four mammals studied. The tissue osmolality was significantly higher than that seen in the same species during in vitro conditions. A villus hyperosmolality was observed also in species which exhibited a net fluid secretion (guinea-pig, rabbit ileum), indicating that the fluid secretion emanated from the intestinal crypts. Based on the results of the present experiments and on observations made in earlier experiments performed on the cat, it is proposed that the villus hyperosmolality is created by a countercurrent multiplier present in the intestinal villus. The hyperosmolar compartment in the villus tissue creates the force that drives fluid from lumen to tissue.