Acute nitric oxide (NO) inhibits angiotensin II (Ang II)-stimulated aldosterone synthesis in zona glomerulosa (ZG) cells. In this study, we investigated the effects of chronic administration of NO on the angiotensin receptor type I (AT1) expression and aldosterone synthesis. ZG cells were treated daily with DETA NONOate (10^-4 M), an NO donor, for 0, 12, 24, 48, 72 and 96 h. Chinese chicken ovary (CHO) cells, stably transfected with the AT1B receptor, were used as a positive control. Western blot analysis indicated that AT1 receptor expression was decreased as a function of time of NO administration in both CHO and ZG cells. Ang II binding to its receptors was determined by radioligand binding. NO treatment of ZG cells for 96 h resulted in a decrease in Ang II binding as compared to control. The B_max was decreased to 1,864 ± 129 fmol/mg protein from 3,157 ± 220 fmol/mg protein (p < 0.005), but the K_d was not changed (1.95 ± 0.22 nM vs 1.88 ± 0.21 nM). The confocal Raman microspectroscopy and immunocytochemistry both confirm that the expression of AT1 receptors in ZG cells decreased with chronic NO administration. In addition, chronic NO administration also decreased the expression of cholesterol side chain cleavage enzyme in ZG cells and inhibited the Ang II- and 25-hydroxycholesterol-stimulated aldosterone synthesis in ZG cells. This study demonstrates that the chronic administration of NO inhibits the aldosterone synthesis in ZG cells by down regulation of the expression of both AT1 receptors and cholesterol side-chain cleavage enzyme.