Adrenaline plays a key role in the control of vasomotor tone; however, the participation of the NO/cGMP pathway in response to -adrenoceptor activation remains controversial. To evaluate the involvement of the endothelium in the vascular response to adrenaline, we assessed the NO production, endothelial NO synthase phosphorylation and tissue accumulation of cGMP in the perfused arterial mesenteric bed of rat. Adrenaline elicited a concentration-dependent increase in NO (EC₅₀ 45.7 pM) which was coupled to cGMP tissue accumulation. Both NO and cGMP production were blocked by either endothelium removal (saponin) or NO synthase inhibition (N-nitro-L-arginine). Blockade of ₁- and ₂-adrenoceptors with 1 µM propranolol or ₃-adrenoceptor with 10 nM SR 59230A displaced rightward the concentration-NO production curve evoked by adrenaline. Selective stimulation of ₁-, ₂- or ₃-adrenoceptors also resulted in NO and cGMP production. Propranolol (1 µM) inhibited the rise in NO induced by isoproterenol or the ₂-adrenoceptor agonists salbutamol, terbutaline or fenoterol. Likewise, 10 nM SR 59230A reduced the effects of the ₃-adrenoceptor agonists BRL 37344, CGP 12177, SR 595611A or pindolol. The NO production induced by adrenaline and BRL 37344 was associated with the activation of the PI3K/Akt pathway and phosphorylation of eNOS in serine 1177. In addition, in anaesthetized rats, bolus administration of isoproterenol, salbutamol or BRL 37344 produced NO-dependent reductions in systolic blood pressure. These findings indicate that ₁-, ₂- and ₃-adrenoceptors are coupled to the NO/cGMP pathway, highlighting the role of the endothelium in the vasomotor action elicited by adrenaline and related -adrenoceptor agonists.