Our objective was to test the hypothesis that fetal urine contains a substance(s) that regulates amniotic fluid volume by altering the rate of intramembranous absorption of amniotic fluid. In late gestation ovine fetuses, amniotic fluid volumes, urine and lung liquid production rates, swallowed volumes and intramembranous volume and solute absorption rates were measured over 2 day periods under control conditions and when urine was removed and continuously replaced at an equal rate with exogenous fluid. Intramembranous volume absorption rate decreased by 40% when urine was replaced with lactated Ringer's solution or lactated Ringer's solution diluted 50% with water. Amniotic fluid volume doubled under both conditions. Analysis of the intramembranous sodium and chloride fluxes suggests that the active but not passive component of intramembranous volume absorption was altered by urine replacement whereas both active and passive components of solute fluxes were altered. We conclude that fetal urine contains an unidentified substance(s) that stimulates active intramembranous transport of amniotic fluid across the amnion into the underlying fetal vasculature and thereby functions as a regulator of amniotic fluid volume.