The addition of ovine prolactin (oPRL) to the fetal side of human term amnion in vitro is associated with a decrease in membrane permeability to tritiated water (THO). As the concentration of oPRL is increased from 2.5 to 20.0 micrograms per milliliter, permeability is progressively impaired. The addition of a specific prolactin receptor antibody completely abolished the effect of oPRL. Also, the addition of Ouabain abolished the effect of oPRL. When an osmolic gradient was created using Dextran-10 on the maternal side of the amnion, the bulk flow of water in control and 3PRL-treated membranes was not significantly different. These findings suggest that PRL acts predominantly on the diffusional flow rather than the bulk flow of water across amnion and that it is a transcellular transport. These studies also represent indirect evidence for the existence of prolactin receptor sites at the level of the amnion.