The objective of this study was to determine whether activation of protein kinase B (PKB) is involved in the production of nitric oxide (NO) induced by cAMP-signal transduction. Mongrel dogs were used for this study. Coronary microvessels were isolated from left ventricular free wall of these dog hearts. Forskolin (an activator of adenylyl cyclase that increases intracellular cAMP level) and 8-bromo-cAMP (a membrane permeable cAMP analogue) were used to stimulate NO release and activation of PKB and endothelial NO synthase (eNOS) in these blood vessels. We found that forskolin and 8-bromo-cAMP increased NO release (quantified using Griess Reaction) from coronary microvessels by 80±6 and 78±11 pmol/mg, respectively (p<0.05 vs. control). Western Blot showed that forskolin elicited a significant increase in eNOS phosphorylation at serine 1177 (Ser1177, a positively regulatory phosphorylation site for eNOS, by 59±11%) and dephosphorylation at threonine 495 (Thr495, a negatively regulatory phosphorylation site of eNOS, by 28±6%), respectively (p<0.05 vs. control). Interestingly, forskolin also increased the phosphorylation of PKB at serine 473 (Ser473, by 49±17%) and threonine 308 (Thr308, by 53±17%), respectively (p<0.05 vs. control, both sites phosphorylation are required for a full activation of PKB). N^w-nitro-L-arginine methyl ester (L-NAME, a NO synthase inhibitor) blocked NO formation, Rp-cAMP [a protein kinase A (PKA) inhibitor] and LY294002 [a PI3-kinase (an activator of PKB) inhibitor] prevented the production of NO, phosphorylation of PKB and eNOS induced by forskolin. Our data clearly show an involvement of PKB activation in cAMP induced NO production. We are reporting for the first time that cAMP-signal transduction stimulates eNOS activation through a PKB-mediated mechanism.