The role of nitric oxide (NO) in the regulation of aldosterone synthesis in the adrenal glomerulosa is not known. In this study, we observed that liberators of NO such as S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (Snp) and spermine nonoate (SNO) could significantly inhibit angiotensin II (AII) and ACTH-induced aldosterone synthesis in isolated rat and cultured human adrenal glomerulosa cells. To evaluate more precisely whether glomerulosa cells express NO synthase, we performed immunoblotting experiments with antibodies specific for the endothelial type ecNO synthase as well as the neuronal NO synthase. This revealed the presence of the ecNO synthase in rat adrenal capsules, in normal and in adenomatous human adrenal glomerulosa tissue, as well as in freshly dispersed rat adrenal glomerulosa cells. Furthermore, on immunohistochemical analysis, rat adrenal glomerulosa cell sections showed strongly positive staining for ecNO synthase. These results suggest that NO may be an important negative modulator of adrenal glomerulosa steroidogenesis.