No studies have specifically addressed whether cAMP can influence NO/cGMP-induced cerebral vasodilation. In this study, we examined whether cAMP can enhance or reduce NO-induced cerebral vasodilation in vivo via interfering with cGMP efflux or through potentiating phosphodiesterase-5 (PDE5)-mediated cGMP breakdown, respectively, in cerebral vascular smooth muscle cells (CVSMCs). To that end, we evaluated, in male rats, the effects of knockdown (via antisense oligodeoxynucleotide [ODN] applications) of the cGMP efflux protein, multidrug resistance protein-5 (MRP5), and PDE5 inhibition, on pial arteriolar NO donor (SNAP)-induced dilations in the absence and presence of cAMP elevations via forskolin. Pial arteriolar diameter changes were measured using well-established protocols in anesthetized rats. In control (missense ODN-treated) rats, forskolin elicited a leftward shift in the SNAP dose-response curves (~50% reduction in the SNAP EC₅₀). However, in MRP5 knockdown rats, cAMP increases were associated with a substantial reduction in SNAP-induced vasodilations (reflected as a significant 35-50% lower maximal response). In the presence of the PDE5 inhibitor, MY-5445, the repression of the NO donor response accompanying forskolin was prevented. These findings suggest that cAMP has opposing effects on NO-stimulated cGMP increases. On the one hand, cAMP limits CVSMC cGMP loss by restricting cGMP efflux. On the other, cAMP appears to enhance PDE5-mediated cGMP breakdown. However, since increased endogenous cAMP seems to potentiate NO/cGMP-induced arteriolar relaxation when MRP5 expression is normal, the effect of cAMP to reduce cGMP efflux appears to predominate over cAMP-stimulation of cGMP hydrolysis.