The present study was undertaken to define the possible role of endogenous ouabain-like substance (EOLS) in controlling renal excretory pattern, in mediating the renal responses to furosemide and in inducing endocrine reactions in furosemide-treated neonates. Ten newborn infants with mean birthweight of 2,752 g and mean gestational age of 37.1 weeks were given furosemide in a dose of 1 mg/kg. Prior to and following furosemide therapy, urine was collected for a period of 12 h and analyzed for creatinine, osmolality, sodium and potassium, as well as for EOLS, arginine vasopressin (AVP), aldosterone and endothelin-1 (ET-1). In response to furosemide administration, urine flow rate and urinary osmolar, sodium and potassium excretion increased significantly, whereas creatinine excretion remained unchanged. Furthermore, following furosemide therapy, urinary excretion of EOLS (148.7 ± 70.8 vs. 200.1 ± 98.1 pg/kg/h) and AVP (19.5 ± 5.4 vs. 27.8 ± 7.8 pg/kg/h) tended to increase, whereas ET-1 (36.0 ± 5.6 vs. 61.4 ± 8.7fmol/kg/h, p < 0.01) and aldosterone (507 ± 120 vs. 751 ± 203 ng/kg/h, p < 0.05) increased significantly. Prior to furosemide, urinary EOLS excretion was found to correlate positively with diuresis (r = 0.80, p < 0.01), sodium (r = 0.91, p < 0.001), creatinine (r = 0.75, p < 0.001), osmolar (r = 0.96, p < 0.001), ET-1 (r = 0.90, p < 0.001) and AVP (r = 0.92, p < 0.001) excretion. After furosemide, urinary EOLS excretion significantly correlated only with diuresis (r = 0.69, p < 0.05), ET-1 (r = 0.77, p < 0.01) and AVP (r = 0.92, p < 0.001). Urinary EOLS proved to be independent of aldosterone excretion irrespective of furosemide administration. It is concluded that urinary EOLS excretion is closely related to neonatal renal excretory pattern and it may have a role in controlling diuresis and natriuresis. The renal response to furosemide appears to be independent of EOLS, although interdependent endocrine reactions can be induced by furosemide which may modulate the production rate and urinary excretion of EOLS.