Previous studies have indicated that 20-hydroxyeicosatetraenoic acid (20-HETE) inhibits Na+ transport in the medullary thick ascending loop of Henle (mTALH), but the mechanisms involved remain uncertain. The present study compared the effects of 20-HETE with those of ouabain and furosemide on intracellular Na⁺ concentration ([Na⁺]_i), Na⁺-K⁺-ATPase activity and ⁸⁶Rb⁺ uptake, an index of Na⁺ transport, in mTALH isolated from rats. Ouabain (2 mM) increased, while furosemide (100 µM) decreased, [Na⁺]_i in the mTALH of rats. Ouabain and furosemide inhibited ⁸⁶Rb⁺ uptake by 91% and 30%, respectively. 20-HETE (1 µM) had a similar effect as ouabain and increased [Na⁺]_i from 19±1 to 30±1 mM. 20-HETE reduced Na⁺-K⁺-ATPase activity by 30% and ⁸⁶Rb⁺ uptake by 37%, but it had no effect on ⁸⁶Rb⁺ uptake or [Na+]i in the mTALH of rats pretreated with ouabain. 20-HETE inhibited 86Rb+ uptake by 12% and increased [Na⁺]_i by 19 mM in mTALH pretreated with furosemide. These findings indicate that 20-HETE secondarily inhibits Na+ transport in the mTALH of the rat, at least, in part by inhibiting the Na⁺-K⁺ -ATPase activity and raising [Na⁺]_i.