Insulin is known to modulate several functions in bovine adrenal cells, including steroidogenesis and growth. However, the role of insulin in regulating aldosterone synthesis in human adrenal glomerulosa cells has not been studied. In the present studies, we have examined the acute and chronic effects of insulin on angiotensin II (ANG II)-induced aldosterone synthesis in cultured normal and adenomatous human adrenal glomerulosa cells. Short-term insulin treatment (1.5 h) resulted in inhibition of ANG II-induced aldosterone synthesis. In contrast, chronic treatment (30 h) resulted in potentiation of ANG II action. The 12-lipoxygenase pathway of arachidonate metabolism has been shown to play a key role in ANG II-induced aldosterone synthesis. The acute inhibitory effects of insulin were in part mediated by inhibition of the 12-lipoxygenase pathway. The chronic stimulatory effect of insulin seemed to be due at least in part to the upregulation of cytochrome P-450 side-chain cleavage enzyme levels. These findings suggest that insulin has a differential temporal effect on ANG II action and therefore may be an important regulator of ANG II-induced aldosterone synthesis in the adrenal.