As evidence continues to accumulate supporting a specific effect of prolactin on the permeability of reflected fetal membranes to tritiated water, no information on the role of decidual prolactin in this process is available. Simulation of in vivo events was conducted with the use of isolated membrane preparations of human amnion or chorioamnion with adherent decidua. In the presence of ovine prolactin in equal concentrations on both sides of human amnion, membrane permeability (cm X 10(-4) X sec-1) was reduced from 1.52 +/- 0.16 to 1.18 +/- 0.07 (mean +/- SEM) from the fetal to the maternal surface. Permeability in the opposite direction was significantly increased from 1.15 +/- 0.10 to 1.39 +/- 0.08. No osmotic effect of ovine prolactin was noted. With the use of chorioamnion with adherent decidua, permeability was significantly reduced from the maternal to the fetal surfaces only when ovine prolactin bathed the maternal surface. These in vitro results may suggest that the accumulation of decidual prolactin at the choriodecidual interface in vivo supports bulk water flow across fetal membrane from the amniotic to the maternal compartments.