Background and PurposeTo analyse the relative contribution of β1‐, β2‐ and β3‐adrenoceptors (Adrb) to vasodilatation in conductance and resistance vessels, assessing the role of cAMP and/or NO/cGMP signalling pathways.Experimental ApproachRat mesenteric resistance artery (MRA) and aorta were used to analyse the Adrb expression by real‐time‐PCR and immunohistochemistry, and for the pharmacological characterization of Adrb‐mediated activity by wire myography and tissue nucleotide accumulation.Key ResultsThe mRNAs and protein for all Adrb were identified in endothelium and/or smooth muscle cells (SMCs) in both vessels. In MRA, Adrb1 signalled through cAMP, Adrb3 through both cAMP and cGMP, but Adrb2, did not activate nucleotide formation; isoprenaline relaxation was inhibited by propranolol (β1, β2), CGP20712A (β1), and SQ22536 (adenylyl cyclase inhibitor), but not by ICI118,551 (β2), SR59230A (β3), ODQ (soluble guanylyl cyclase inhibitor), L‐NAME or endothelium removal. In aorta, Adrb1 signalled through cAMP, while β2‐ and β3‐subtypes through cGMP; isoprenaline relaxation was inhibited by propranolol, ICI118,551, ODQ, L‐NAME, and to a lesser extent, by endothelium removal. CL316243 (β3‐agonist) relaxed aorta, but not MRA.Conclusion and ImplicationDespite all three Adrb subtypes being found in both vessels, Adrb1, located in SMCs and acting through the adenylyl cyclase/cAMP pathway, are primarily responsible for vasodilatation in MRA. However, Adrb‐mediated vasodilatation in aorta is driven by endothelial Adrb2 and Adrb3, but also by the Adrb2 present in SMCs, and is coupled to the NO/cGMP pathway. These results could help to understand the different physiological roles played by Adrb signalling in regulating conductance and resistance vessels.