Experiments conducted in vitro suggest that high osmolality stimulates endothelin production and release by renal tubular epithelial cells. Whether hyperosmotic solutions exert similar effects in vivo is unknown. Therefore we tested the hypothesis that increasing renal medullary osmolality enhances urinary excretion of endothelin in anesthetized rats. Isosmotic NaCl (284 mOsmol/kg H₂O) was infused either intravenously (1.5 ml/h) or into the renal medullary interstitium (0.5 ml/h) during a 1 h equilibration period and 30 min baseline urine collection period, followed by either isosmotic or hyperosmotic NaCl (921 or 1664 mOsmol/kg H₂O i.v.; 1714 mOsmol/kg H₂O into renal medulla) for two further 30 min periods. Compared to isosmotic NaCl, infusion of hyperosmotic NaCl into the renal medulla significantly increased the endothelin excretion rate (P < 0.05; from 0.30 ± 0.02 to 0.49 ± 0.03 fmol/min). Intravenous infusion of hyperosmotic NaCl also significantly increased endothelin excretion rate in a concentration-dependent manner (from 0.79 ± 0.07 to 1.77 ± 0.16 fmol/min and 0.59 ± 0.04 to 1.11 ± 0.08 fmol/min for 1664 and 921 mOsmol/kg H₂O respectively). To differentiate between effects of osmolality and NaCl, similar experiments were performed using mannitol solutions. Compared to isosmotic mannitol, medullary interstitial infusion of hyperosmotic mannitol (1820 mOsmol/kg H₂O) significantly increased endothelin excretion rate (P < 0.05; from 0.54 ± 0.03 to 0.94 ± 0.12 fmol/min). Thus exposing the renal medulla to hyperosmotic concentrations of either NaCl or mannitol stimulates endothelin release in vivo, consistent with medullary osmolality being an important regulator of renal endothelin synthesis.