High concentrations of digoxin-like immunoreactive substance (DLIS) have been identified in amniotic fluid and in blood during pregnancy. In this study, urine from healthy women was analyzed for DLIS during pregnancy and following delivery, and levels were related to various clinical and urinary findings. Urinary DLIS increased progressively during pregnancy, having a linear relation to gestational week (DLIS = 0.032 week + 0.46, Syx = 0.52, r = 0.46, p less than 0.01), and fell to nonpregnant values during the first postpartum day. The urinary DLIS level correlated with urinary creatinine (r = 0.50, p less than 0.01) and urinary calcium (r = 0.56, p less than 0.01) levels. When adjusted for the gestational week, the urinary DLIS level also correlated with urinary potassium (r = 0.48, p less than 0.01), creatinine (r = 0.55, p less than 0.01), and magnesium (r = 0.55, p less than 0.01) levels. After adjustment for the urinary creatinine level, only the relation of urinary DLIS and urinary calcium levels remained significant (r = 0.44, p less than 0.05). The urinary DLIS level was not found to be related to systemic blood pressure. The ratios of sodium to potassium (r = -0.43, p less than 0.05) and calcium to magnesium (r = 0.37, p less than 0.05) were also related to gestational week but were independent of the urinary DLIS level. Thus, urinary DLIS increases progressively during pregnancy and falls to nonpregnant values on the first postpartum day, the urinary DLIS level can be related to urinary creatinine, urinary potassium, and urinary magnesium levels, and when adjusted for the urinary creatinine level, the urinary calcium level relates independently to the urinary DLIS level.