In addition to β₁-, β₂-adrenoceptors, the existence of vasodilatory, propranolol-insensitive atypical β-adrenoceptors has been suggested in rat and human vessels. They are activated by non-conventional partial β-adrenoceptors agonists – cyanopindolol (CYP) and CGP 12 177 (CGP) in an endothelium-dependent manner [1, 2]. The aim of the present study was to examine the endothelium-dependent relaxant mechanism of CYP and CGP in the rat mesenteric artery. For comparison, the β₂-adrenoceptor agonist fenoterol (FEN) was used. Male Wistar rats (8-week-old/ 280-350g) were killed by decapitation. Mesenteric arteries pre-constricted with phenylephrine (1μM) were relaxed in a concentration-dependent fashion by CYP (pD₂=5.29±0.04; E_max=105.2±1.3; n=12), CGP (pD₂=4.5±0.08; E_max=103.9±1.5; n=18) and FEN (pD₂=4.42±0.1; E_max=98.4±3.1; n=11). Relaxations were either insensitive (CYP, CGP) or sensitive (FEN) to propranolol (0.3 μM). In endothelium-intact vessels the inhibitor of nitric oxide synthase L-N^G-nitroarginine methyl ester (L-NAME; 300 μM) diminished the relaxation evoked by FEN, but not by CYP and CGP. Vasodilatory effects of all agonists were insensitive to the inhibitor of cylooxygenase indomethacin (INDO; 10 μM) but they were inhibited by tetraethylammonium (TEA; 300 μM) and 4-amidopyridine (4-AP; 1mM), calcium activated (K_Ca) and voltage-gated (K_V) potassium channels blockers, respectively. The blocker of ATP-sensitive potassium channels glibenclamide (10 μM) reduced the relaxation elicited by FEN but not by CYP and CGP. All agonists were less potent in arteries pre-constricted with 60 mM KCl compared to phenylephrine-pre-constricted vessels. Combined pre-incubation with L-NAME, INDO, Ba²⁺ (the inhibitor of inward rectifying potassium channel; 30 μM) and ouabain (the inhibitor of Na⁺/K⁺-ATP-ase; 1mM) increased the basic level of vessel tone and attenuated the vasorelaxant response to CYP. Thus, our results demonstrate that CYP and CGP evoked an endothelium-dependent relaxation via activation of K_Ca and K_V, but not K_ATP, perhaps via propranolol-insensitive β-adrenoceptors.