ABSTRACT.An endogenous digoxinlike immunoreactive substance(s) (DLIS) exists in the serum of premature and full term infants not receiving digoxin. We followed serum changes in DLIS concentration sequentially over the first 14 postnatal days in 24 premature neonates who did not receive digoxin in the intensive care nursery. All infants had measurable levels (>0.6 ng/ml) of DLIS in their serum. There was a distinct peak in DLIS concentration in 19 of 24 infants occurring at 4 ± 1.6 (SD) days after birth (range, 1–8 days). No peak was found in five infants. The peak serum level of DLIS obtained in the first 8 days of life was negatively correlated with gestational age and birth weight.DLIS levels in amniotic fluid remained constant from 16 to 33 weeks of gestation but rose from 33 wk to term. DLIS concentrations in umbilical artery, umbilical vein, and maternal serum at normal full term delivery suggested that DLIS was of fetal origin. DLIS and digoxin concentrations are additive when present in the same serum sample if measured by standard radioimmunoassay methods.