Nitric oxide (NO) is known to be critical endogenous regulator of vascular cells function. NO donors are widely used as pharmacological tool to understand the physiological effect of NO and to treat cardiovascular diseases. The major clinical benefit of the organic nitrates like Nitroglycerin (GTN) is attributed to their potent venodilator effect. However, the chronic use of GTN is limited by nitrate tolerance that is characterized by rapid loss of its effects or cross-tolerance to other vasodilator. This study aimed to verify if the new NO donor synthesized in our laboratory (RuBPY) induces in vitro tolerance and cross-tolerance to acetylcholine (ACh) and sodium nitroprusside (SNP) in rat cava vein. All experimental protocols were performed in accordance to the Ethical Principles in Animal Experimentation adopted by the Brazilian College of Animal Experimentation (COBEA) and were approved by the Ethics Committee on Animal Use (CEUA) of the University of Sao Paulo (Protocol 11.828.532). Rats (200 to 230g, n=25) were killed under anesthesia with isofluorane (500 µL/animal).The cava vein was isolated and used to vascular reactivity and western blot (WB) studies. The vasodilatation induced by RuBPY and GTN were compared by the maximum effect (ME) and potency (pD2). In vitro tolerance was induced by incubation of the veins for 10, 30 or 60 min with RuBPY (2 µM or 10 µM) or GTN (4 µM or 100 µM). In vitro cross-tolerance to ACh and SNP was induced by the veins pre-exposure for 60 min to RuBPY (2 µM) or GTN (4 µM). The eNOS phosphorylated in the activation site (Ser1177), the inactivation site (Thr495) and the ratio of active eNOS dimers to inactive eNOS monomers was evaluated by WB. Our results demonstrated that RuBPY induced greater relaxation (ME: 92.8 ± 4.4%, n=7; P<0.05) than GTN (ME: 75.3 ± 3.7%, n=6). Previous exposure for 10 min to RuBPY (2 µM or 10 µM) or GTN (4 µM or 100 µM) did not induce tolerance. Pre-exposure for 30 min to 2 µM or 10 µM RuBPY did not alter the relaxation to RuBPY. However, pre-exposure for 30 min to 4 µM or 100 µM GTN reduced the relaxation to GTN (ME: 45.4 ± 2.2%, n= 6; P<0.05 and ME: 39.2 ± 1.4%, n= 6; P<0.05), respectively. Pre-exposure for 60 min to RuBPY reduced the relaxation in the concentrations of 2 µM (ME: 48.0 ± 2.3%, n= 7; P<0.05) and 10 µM (ME: 30.1±1.2%, n= 7; P<0.05). Pre-exposure to RuBPY or GTN did not reduce the relaxation to SNP. Cross-tolerance to ACh was induced only with GTN. The ME induced by ACh was reduced from 100.3 ± 5.3% to 75.1 ± 4.2%, n=7; P<0.05). Although RuBPY and GTN phosphorylated eNOS in the inhibitory site (Thr495), they did not modify the eNOS dimers/monomers ratio. Taken together, our results show that RuBPY takes more time (60 min) than GTN (30 min) to induce tolerance and it does not induce cross-tolerance with acetylcholine.