Introduction: Perivascular adipose tissue (PVAT) exerts an anticontractile effect. Studies using various vasoconstrictors suggest that this may be dependent on adrenergic stimulation and the subsequent release of nitric oxide. This has not previously been investigated in PVAT but β3-adrenoceptors are known to be the predominant subtype mediating lipolysis in rodent adipocytes. Design and methods: The effects of PVAT on the contractility of isolated Wistar rat mesenteric arteries were investigated using wire myography. All procedures were performed in accordance with Scientific Procedures Act (1986). Concentration-response curves to noradrenaline (1×10-5 – 3×10-9 mol.l-1) or phenylephrine (1×10-5 – 3×10-9mol.l-1) were generated in the presence and absence of the β3-adrenergic agonist, CL-316,243 (10 µmol.l-1). In addition, a Griess reagent kit was used to determine NO production from adipose tissue surrounding the mesenteric artery in response to adrenergic agonist stimulation +/- the nitric oxide synthase inhibitor, L-NMMA (100 µmol.l-1). Results and summary: The vasoconstrictor response to noradrenaline was reduced in the presence of PVAT through an endothelium-dependent mechanism involving activation of vascular smooth muscle K+ channels (endo: PVAT vs no PVAT P<0.01, n = 12, no endo: PVAT vs no PVAT P=0.54, n=12). A reduced anticontractile effect was observed in response to phenylephrine (P=0.45, n =12), which acts mainly via α-adrenergic receptors, suggesting that the anticontractile effect of PVAT is dependent on β-adrenergic stimulation. Moreover, in vessels with intact PVAT but lacking endothelium, the presence of CL-316,243 caused a reduction in the vasoconstrictor effect of phenylephrine (P<0.05, n=6) whereas the vasoconstrictor response to noradrenaline was unaltered reaffirming the role of β-adrenergic stimulation in the anticontractile effect of PVAT. Adrenergic stimulation of adipose tissue (devoid of vessels) with noradrenaline and CL-316,243, but not phenylephrine, was associated with a significant increase in nitric oxide production (P<0.05, n=5), which was inhibited by the presence of L-NMMA. The similarity in response to noradrenaline and CL-316,243 suggests that β3-adrenoceptors are the main β-adrenergic subtype mediating this response. Overall, this study indicates a role for β-adrenergic stimulation and subsequent nitric oxide production in the anticontractile action of PVAT.