This study investigated the effects of two NO-releasing agents, diethylenetriamine-NO (deta-NO) and sodium nitroprusside (SNP), on basal, ACTH-, and angiotensin II (AngII)-stimulated aldosterone production in glomerulosa cells from bovine adrenal gland. NO donors inhibited basal and ACTH- or AngII-stimulated aldosterone synthesis in a concentration-dependent manner. Deta-NO and SNP also provoked a concentration-dependent stimulation of cGMP production. However, cGMP was not responsible for the inhibition of aldosterone secretion, because a cGMP analog did not reproduce the inhibitory effect. Moreover, soluble guanylyl cyclase or protein kinase G inhibitors did not revert the inhibitory effect of NO on aldosterone production. NO donors did not modify ACTH-stimulated cAMP production or AngII-stimulated PLC activity stimulation, but inhibited 22[R] hydroxycholesterol- or pregnenolone-stimulated aldosteronogenesis. NO can be synthesized in bovine glomerulosa cells because nitrite production was determined and characterization of NOS activity was also performed. Nitrite accumulation was not modified in the presence of ACTH, AngII, or other factors used to induce iNOS. NOS activity that showed a Michaelis-Menten kinetic was NADPH- and calcium-dependent and was inhibited by two competitive inhibitors, L-NAME and L-NMMA. These results show that NO inhibits aldosterone production in glomerulosa cells acting on P450scc and other P450-dependent steroidogenic enzymes, and these cells display NOS activity suggesting that NO can be produced by constitutive NOS isozymes.