We report the presence of an immunoreactive digoxin-like substance in blood from third trimester pregnant women. The sera from 51 women in the third trimester of pregnancy were analyzed by 4 commercially available digoxin RIAs. None of these patients was receiving digoxin. Digoxinimmunoreactivity was detected in all patients by 3 of 4 assays. The measured values, in nanograms per ml digoxin equivalent, were (mean ± SD): method A, 0.27± 0.05; method B, 0.28 ± 0.07; method C, 0.01 ± 0.01; and method D, 0.15 ± 0.06. Method B measured values greater than 0.50 ng/ml in sera from 5 patients. Digoxin immunoactivity was not detectable 24 h postpartum, suggesting a half-life in serum of 6 h or less. Exogenous digoxin added to these serum samples resulted in quantitatively additive increments above the endogenous measured levels. Three of 4 digoxin RIAs did not distinguish between true digoxin and the endogenous substance present in the sera of thirdtrimester pregnant patients. Preliminary evidence suggests that the endogenous digoxin immunoactivity is not due to elevation of levels of major knownsteroids in the blood of these women. Clinical management of women requiring digoxin therapy during pregnancy, therefore, is complicated by the inability to assume the same therapeutic range of digoxin in serum during the third trimester of pregnancy as in adult nonpregnant individuals. (J Clin Endocrinol Metab58: 748, 1984)