Radioimmunologic techniques were used to determine 2 primary bile acids (cholic and chenodeoxycholic acid) and 1 secondary bile acid (deoxycholic acid) from human amniotic fluid of healthy pregnant women and from patients with diabetes, toxemia, or intrahepatic cholestasis during pregnancy. In general, the mean bile acid concentrations in the amniotic fluid were very similar to those in the serum, although in paired samples from individual patients these 2 values did not correlate significantly. Very high levels of the 2 primary bile acids were measured from the amniotic fluid of patients with intrahepatic cholestasis. The mean values were about 70 times higher than those in the controls. Amniotic fluid cholic acid content was slightly elevated in diabetic and toxemic patients, too. Deoxycholic acid was consistently found in the amniotic fluid specimens, but there was no change in its concentration among the various groups. In this limited series of patients, no significant correlation was found between the bile acid concentrations in the amniotic fluid and signs of fetal distress at the time of amniocentesis, although the lowest maternal serum estriol and human placental lactogen values were associated with the highest amniotic fluid bile acid concentrations. The condition of the newborn infants did not correlate with amniotic fluid bile acid concentrations in any of the patient groups studied. It thus appears that high amniotic fluid bile acid content present a threat to the fetus, but further studies are needed to clarify this point.