Cytoplasmic microtubules are important in many cellular homeostatic processes in the cell. They regulate cell shape and movement, as well as serve as a network by which vesicles and membrane-bound organelles can travel. Lately, there are many studies demonstrating that microtubules are involved in the regulation of intracellular signaling, therefore, affecting vascular reactivity. In this study, we tested the hypothesis that microtubule disruption attenuates agonist-induced endothelium-dependent vasodilation. Isolated mesenteric arterial bed from normotensive rats was pre-constricted with phenylephrine and dose-response curves for histamine, acetylcholine, sodium nitroprusside and pinacidil were performed before and after incubation with nocodazole or colchicine. Treatment of the vascular beds with nocodazole or colchicine significantly attenuated histamine relaxation, but did not change the acetylcholine, sodium nitroprusside or pinacidil induced vasorelaxation. Nocodazole did not cause an additional attenuation of the histamine-mediated dilation in mesenteric vessels in the presence of L-NAME, high extracellular potassium or potassium channels blockers. These data suggest that disruption of microtubules affects an essential endothelial component for the histamine-mediated vasodilation in the mesenteric arterial bed. The mechanism(s) involved in this effect might be related to an impairment of endothelial nitric oxide synthesis, which might not be as important for the acetylcholine as for histamine vasodilator response in rat mesenteric vessels. These results demonstrate an importance of the microtubular system for the endothelium-dependent nitric oxide mediated smooth muscle relaxation.