We have investigated the role of cAMP in NO- and prostanoid-independent relaxations that are widely attributed to an endothelium-derived hyperpolarizing factor (EDHF). Under control conditions EDHF-type relaxations evoked by acetylcholine (ACh) in rabbit iliac arteries were transient, but in the presence of the cAMP phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) or the cell permeant cAMP analog 8-bromo-cAMP, relaxations became sustained with their maxima potentiated approximately 2-fold. Relaxation was associated with transient approximately 1.5-fold elevations in smooth muscle cAMP levels with both mechanical and nucleotide responses being abolished by interrupting gap junctional communication with the connexin-mimetic peptide Gap 27 and by endothelial denudation. However, IBMX induced a sustained endothelium-independent approximately 2-fold rise in cAMP levels, which was not further amplified by ACh, suggesting that the contribution of cAMP to the EDHF phenomenon is permissive. After selective loading of the endothelium with calcein AM, direct transfer of dye from the endothelium to the media was enhanced by IBMX or 8-bromo-cAMP, but not by 8-bromo-cGMP, whereas Gap 27 promoted sequestration within the intima. ACh-induced hyperpolarizations of subintimal smooth muscle in arterial strips with intact endothelium were abolished by Gap 27 and the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine but were unaffected by IBMX. By contrast, in strips partially denuded of endothelium, IBMX enhanced the transmission of hyperpolarization from the endothelium to remote smooth muscle cells. These findings support the hypothesis that endothelial hyperpolarization underpins the EDHF phenomenon, with cAMP governing subsequent electrotonic signaling via both myoendothelial and homocellular smooth muscle gap junctions.