Cyclic AMP-stimulating agents are powerful vasodilators, but our knowledge of the signal transduction mechanisms of these agents, particularly in human arteries, is limited. We now report direct molecular effects of prostaglandin E<sub>2</sub> (PGE<sub>2</sub>) on cultured human coronary artery smooth muscle cells (HCASMC). Patch-clamp studies revealed that 10 µ M PGE<sub>2</sub> opens a high-conductance (∼200 pS), calcium-stimulated potassium (BK<sub>Ca</sub>) channel in intact HCASMC. In contrast, PGE<sub>2</sub> had no direct effect on channels in cell-free patches, indicating involvement of a soluble second messenger. Enzyme immunoassay demonstrated that PGE<sub>2</sub> enhances production of cAMP in HCASMC, but does not increase [cGMP]. Furthermore, forskolin, CPT-cAMP, or CPT-cGMP mimicked the stimulatory effect of PGE<sub>2</sub> on BK<sub>Ca</sub> channel activity. Interestingly, the response to PGE<sub>2</sub> was unaffected by inhibiting the cAMP-dependent protein kinase, but was antagonized by inhibitors of the cGMP-dependent protein kinase (PKG). Furthermore, cAMP-stimulated PKG activity mimicked the effect of PGE<sub>2</sub>. These studies suggest a novel PGE<sub>2</sub> action in human arteries: opening of BK<sub>Ca</sub> channels via cAMP cross-activation of PKG in HCASMC. It is proposed that this signaling mechanism may mediate the vasodilatory response to cAMP-dependent agents in the human coronary and other vascular beds.