The influence of administration of ovine prolactin in vivo on intestinal fluid and ion transport in vitro was investigated using intact and hypophysectomized male rats. Prolactin administration significantly stimulated fluid, sodium,potassium, calcium, magnesium and chloride transport across everted jejunal sacs. The last two ions were affected less than the others. Hypophysectomy caused a significant decrease in fluid and sodium absorption, but prolactin treatment for 2 days restored normal absorption rates but not uniformly in all sacs. Prolactin action on fluid and sodium absorption showed a dose-dependent tendency, maximal stimulation resulting from administration of 1.0 to 2.0 mg prolactin daily; higher doses failed to elicit significant response. The stimulatory action of prolactin was inhibited by a simultaneous administration of vasopressin which when given alone had no effect on intestinal absorption. In the absence of glucose or in the presence of phlorizin, fluid transport was inhibited, the reduction being more dramatic in the presence of phlorizin. Similarly, either application of ouabain or partial replacement of sodium with isotonic choline chloride reduced fluid transport. Although these in vitro treatments nullified the stimulatory effects of prolactin, only phlorizin and ouabain significantly decreased sodium transport. These results suggest that the effects of prolactin on intestinal transport may be dependent on increased movement of sodium.