Abstract--We have reported recently that the renal angiotensin II type 2 (AT₂) receptors are upregulated and involved in promoting natriuresis/diuresis in obese but not in lean Zucker rats. In the present study, we tested the hypothesis that there is an enhanced AT₂ receptor signaling via NO/cGMP pathway leading to greater inhibition of the Na⁺, K⁺-ATPase (NKA) activity in the proximal tubules (PT) of obese rather than lean Zucker rats. The AT₂ agonist CGP42112 (0.1 to 100 nmol/L) inhibited (33% at 100 nmol/L) the NKA activity in the PTs of obese but not in lean Zucker rats. The AT₂ antagonist PD123319 (1 µmol/L), not the angiotensin II type 1 antagonist losartan (1 µmol/L), significantly diminished the CGP42112-induced inhibition of the NKA activity in obese rats. The AT₂ agonist (10 nmol/L)-induced NKA inhibition was abolished by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10 µmol/L), the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (100 µmol/L), and the protein kinase G inhibitor K1388 (2 µmole/L). CGP42112 (10 nmol/L) caused an increase in serine phosphorylation of NKA 1-subunit in PT of obese rats. Measurement of cGMP and NO revealed that CGP42112 (0.1 to 100 nmol/L) increased cGMP and NO accumulation in the PTs of obese but not lean rats. The CGP42112-induced stimulation of NO and cGMP was blocked by PD123319 (1 µmol/L), N^G-nitro-L-arginine methyl ester (100 µmol/L), and 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10 µmol/L) but not by losartan (1 µmol/L). The data suggest that the AT₂ receptor activation via stimulation of the NO/cGMP/protein kinase G pathway directly inhibits the tubular NKA activity that provides as a mechanism responsible for the AT₂ receptor-mediated natriuresis in obese but not in lean Zucker rats.