Using four different digoxin kits, it was disclosed that the majority of various samples including amniotic fluid, cord blood, and serum from neonates contained substantial levels of digoxin-like immunoreactive substance. The differences in data seemed to be due to the range of epitopes which are recognized by antidigoxin antiserum. The day-to-day studies on sera serially obtained from infants at birth to 48 days old revealed that the level of the substance (0.31 ± 0.12 ng/ml) in sera of the 1-dayold neonates rapidly declined to the level of 0.1 ng/ml by the 2nd postnatal wk and thereafter gradually declined. The immunological specificity and accuracy of the detection of digoxin-like immunoreactive substance was confirmed by a sample dilution test, a recovery test for standard digoxin, and an absorption test with antidigoxin antiserum. The amniotic fluid and cord blood also contained four to eight times more of a digitoxin-like immunoreactive substance than they did digoxin-like immunoreactive substance. A significant correlation was observed between the levels of digoxin-like immunoreactive substance and of digitoxin-like immunoreactive substance (p<0.01).