The nitrite anion (NO2-) can be the major source of intravascular and tissue storage of nitric oxide (NO), important modulator of vascular tone and blood pressure. The NO donor RUBPY synthesized by our group, releases NO inside the vascular smooth muscle cell in a tissue dependent manner. Long-term treatment with organic nitrates like nitroglycerin, leads to the development of tolerance characterized by the rapid loss of vasodilator effects. Tolerance is a multifactorial process. It may be due to endothelial dysfunction that involves increased production of vascular reactive oxygen species (ROS), decreased activity of soluble guanylyl-cyclase and increased phosphodiesterase expression and activity. This study aimed to investigate the in vitro tolerance to the pre-exposure of intact endothelium aorta (e+) or denuded aorta (e-) with RuBPY (EC100:10 μM). It was also evaluate if the compound releases NO in the endothelial cells in the incubation time studied. The aortic rings were isolated and used for the functional studies. Aortic rings (e+/e-) were contracted with phenylephrine, and after reaching a stable and maintained contraction, RuBPY (3 nM-5 μM) was added to the organ bath. Experiments were conducted after pre-exposure to RuBPY (tolerance) or in the absence of RuBPY (control). The maximum effect (ME) and potency (pD2) of RuBPY in inducing relaxation were evaluated. Endothelial cells were incubated with the fluorescent dye DAF-2/DA (10 μM) to quantifying the cytosolic NO and DHE (2.5 μM) to quantifying ROS. The intensity of fluorescence was detected in the absence or after exposure to RuBPY at different times (5-60min). This study was approved by the Ethical Animal Committee of the University of São Paulo (2012.1.134.53.12). The compound RuBPY induced concentration-dependent relaxation in e+ aortas (pD2:7.81±0.18; ME:101.6± 1.4%, n=7) and e- (pD2:7.54±0.13; ME:103.4±0.4%, n=6). The incubation with 10 μM RuBPY for 5 min followed by 20 min of washout did not affect the ME induced by the compound in e+ aorta (pD2:7.99±0.18; ME:98.5 ±1.6%, n=5) or in e- aortas (pD2:7.89±0.18, ME:100.0 ±0.4%, n=6). Pre-exposure for 10 min with RuBPY in e+ aortas did not alter the relaxation to RuBPY compared to respective control. However, in e- aortas the relaxing effect of RuBPY was potentiated (pD2:7.37±0.12, ME:101.5±1.0%, n=5, P<0.05). Pre-exposure for 30 min with RuBPY did not change the relaxation to RuBPY in e- aortas as compared to the control. RuBPY did not release NO and ROS in the endothelial cells up to 60 min incubation. Taking together, our results demonstrate that the new NO donor RuBPY does not induce tolerance in rat aorta, which is probably due to the lack effect of RuBPY on the endothelial cells.