The present study was aimed at investigating the role of endogenous nitric oxide (NO) in regulating Na,K-ATPase activity in the kidney. The expression of alpha-1 and beta-1 subunits; and the enzymatic activity of Na,K-ATPase were determined in the kidney of rats treated with an NO synthase inhibitor, N(G)-nitro- l -arginine methyl ester (l -NAME). Following the treatment with l -NAME in the drinking water for 4 weeks, Na,K-ATPase activity was increased while tissue nitrite/nitrate levels were decreased in the kidney. Supplementation with l -arginine prevented the l -NAME-induced changes. The expression of either alpha-1 or beta-1 subunit protein of Na,K-ATPase, assessed by Western blot analysis, was not affected by l -NAME-treatment. An acute in vitro treatment of the kidney with l -NAME also caused an increase of Na,K-ATPase activity; which was again prevented by cotreatment with l -arginine. On the contrary, treatment with sodium nitroprusside significantly decreased Na,K-ATPase activity. These results suggest that the endogenous NO plays a direct inhibitory role on Na,K-ATPase activity in the kidney. 2000 Academic Press@p$hr Copyright 2000 Academic Press.