This study investigates the effect of water deprivation on the expression of ANP_1-28 binding sites in rat kidney. Water deprivation increased the B_max of glomerular binding sites for ANP_1-28 and CNP_1-22 without modifying their affinity, an effect that was prevented in the presence of C-ANF, suggesting that NPR-C binding sites might be enhanced. Our results indicate that ANP_1-28, CNP_1-22 and C-ANF inhibit cAMP synthesis directly stimulated by forskolin or by the physiological agonists histamine and 5-hydroxytryptamine . The inhibitory effect was found to be significantly greater in water deprived rats than in controls. Our observations suggests that this effect must be attributed to the 67 kDa NPR-C-like protein, since the 67 and 77 kDa NPR-C-like proteins show high and low affinities for CNP_1-22, respectively, and the enhanced inhibitory effect of CNP on cAMP generation in water deprived rats was detected at subnanomolar concentrations. In addition, using affinity cross-linking studies we have observed that water deprivation increases the expression of the 67 kDa NPR-C-like protein, and HS-142, which binds to NPR-A and the 77 kDa NPR-C-like but not the 67 kDa protein, reduced ligand internalization without affecting cAMP inhibition by ANP_1-28. Finally, we have found that ligand binding to the 67 kDa NPR-C-like protein is reduced by GTPS, suggesting that this receptor is associated with a G protein in renal glomeruli. The enhanced inhibitory role of natriuretic peptides on cAMP synthesis induced by water deprivation may influence glomerular function in the rat kidney.