Adrenal blood flow is coupled to adrenal hormone secretion. Adrenocorticotropic hormone (ACTH) increases adrenal blood flow and stimulates the secretion of aldosterone and cortisol in vivo. However, ACTH does not alter vascular tone of isolated adrenal cortical arteries. Mechanisms underlying this discrepancy remain unsolved. The present study examined the effect of zona glomerulosa (ZG) cells on cortical arterial tone. ZG cells (10⁵-10⁷ cells) and ZG cell conditioned medium relaxed preconstricted adrenal arteries (maximal relaxations = 79±4% and 66±4%, respectively). In adrenal arteries coincubated with a small number of ZG cells (0.5-1x10⁶), ACTH (10^-12-10^-8 M) induced concentration-dependent relaxations (maximal relaxation = 67±4%). Similarly, ACTH (10^-8 M) dilated (55±10%) perfused arteries embedded in adrenal cortical slices. ZG cell-dependent relaxations to ACTH were endothelium-independent and inhibited by high extracellular K⁺ (60 mM), the K⁺ channel blocker, iberiotoxin (100 nM), the cytochrome P450 inhibitors SKF 525A (10 µM) and miconazole (10 µM) and the epoxyeicosatrienoic acid (EET) antagonist 14,15-EEZE (2 µM). Four EET regioisomers were identified in ZG cell conditioned media. EET production was stimulated by ACTH. We conclude that ZG cells release EETs and this release is stimulated by ACTH. Interaction of endocrine and vascular cells represents a mechanism for regulating adrenal blood flow and couples steroidogenesis to increased blood flow.