To examine the effect of prolactin (PRL) on transplacental water flow, we infused mannitol (500 ml; 20% solution) over 10 min into five chronically catheterized ewes (121-134 days' gestation), producing a peak maternal plasma osmolality by 10 min and fetal osmolality by 20 min. One day before or after, an identical amount of mannitol was infused into the same ewe during the 2nd h of a 2-h infusion of PRL (40 ± 2.2 µg/h) into a fetal leg vein. Mean (± SE) fetal plasma PRL levels were 6.9 ± 3.2 ng/ml at baseline. Steady state fetal PRL levels were 17.7 ± 7.4 ng/ml during PRL infusion. Maternal mannitol infused without administration of PRL to the fetus evoked a rise in fetal plasma osmolality similar to that following maternal mannitol during PRL administration to the fetus. Thus, as shown previously, PRL affects water permeability across the membranous chorioamnion, whereas results of the present study indicate that the hormone does not affect water transfer across the ovine chorionic villi (placenta).