We have recently demonstrated that endothelial β₂ adrenergic receptors (β₂AR) regulate eNOS activity and consequently vascular tone, through means of PKB/AKT. In this work we explored the signal transduction pathway leading to AKT/eNOS activation in endothelial cells (EC). Using pharmacological and molecular inhibitors both in cultured EC cells and in ex vivo rat carotid preparations, we found that G_i coupling of the β₂AR is needed for AKT activation and vasorelaxation. Since endothelial activation is sensitive to pertussis toxin but not to G_iβγ inhibition by βARKct, we conclude that G_αi mediates βAR induced AKT activation. Downstream, βAR signalling requires the soluble tyrosine kinase SRC, as both in cultured EC and rat carotid, the mutant dominant negative of SRC prevent β₂AR induced endothelial activation and vasodilation. In EC, G_αi directly interacts with SRC and this interaction leads to SRC activation and phosphorylation in a manner that is regulated by β₂AR stimulation. We propose a novel signal transduction pathway for β₂AR stimulation trough G_αi and SRC, leading to activation of AKT.