Micropuncture studies of the distal nephron and measurements of Na,K-ATPase activity in microdissected collecting tubules have suggested that renal retention of sodium in puromycin aminonucleoside (PAN) nephrotic rats originates in the collecting duct. The present study demonstrated this hypothesis by in vitro microperfusion and showed that amiloride was able to restore sodium balance. Indeed, isolated perfused cortical collecting ducts from PAN-treated rats exhibited an abnormally high transepithelial sodium reabsorption that was abolished by amiloride, and in vivo administration of amiloride fully prevented decreased urinary sodium excretion and positive sodium balance in nephrotic rats. As expected from the aldosterone independence of Na(+) retention in PAN nephrotic rats, blockade of aldosterone receptor by potassium canrenoate did not alter urinary Na(+) excretion, Na(+) balance, or ascites formation in PAN nephrotic rats.