Although fetal urine output and swallowing are major contributors to amniotic fluid regulation, other pathways for fluid movement must be involved in the regulation of amniotic fluid volume because many studies report fetal urine output to be greater than swallowing. This study was designed to examine the possibility of fluid transfer between the amniotic cavity and the fetal blood that perfuses the fetal membranes and surface of the placenta in the ovine fetus. We injected warmed distilled water into the amniotic fluid in three groups of chronically catheterized fetal sheep. In normal fetuses, there was rapid absorption of the water into the fetal circulation, resulting in highly significant decreases in fetal osmolality, plasma electrolytes, and heart rate as well as increases in arterial pressure and fetal hemolysis. Concomitantly, there was a small, delayed fall in maternal osmolality. In a second group of fetuses with ligated esophagi, these same responses occurred except that the fetal osmolality and electrolyte changes occurred earlier and were significantly greater. In a third group of fetuses killed just before the water injection, maternal osmolality was unchanged. These data suggest the intramembranous pathway as a major route of amniotic fluid absorption in the ovine fetus. In addition, esophageal ligation appears to augment the conductance of this pathway, as evidenced by a significantly greater estimated filtration coefficient and rate of water absorption in the ligated animals than in controls. Finally, the transmembranous pathway directly to the mother does not appear to be a major route.