Nitric oxide (NO) donors like nitroglycerin (GTN) are widely used as pharmacological tool to understand the physiological effect of NO, and in the treatment of cardiovascular diseases. The major therapeutic limitation of GTN is the tolerance that is characterized by rapid loss of its effects in long-term administration, or cross-tolerance to other vasodilator, which cause(s) is still not clear. It is endothelium-dependent and the uncoupling endothelial NO-synthase (eNOS) could contribute to the tolerance. The major clinical benefit of organic nitrates, including the GTN has been attributed to their potent venodilator effect. This study aimed to verify if the new NO donor synthesized in our laboratory (RuBPY) induces in vitro tolerance and cross-tolerance to acetylcholine and sodium nitroprusside (SNP) in rat cava vein. We compared the effects of RuBPY with GTN in relation to the maximum effect (ME) and potency (pD2). In vitro tolerance was induced by incubation for 10, 30 or 60 min with RuBPY (2 µM or 10 µM) or GTN (4 µM or 0.1mM). In vitro cross-tolerance to acetylcholine and SNP was induced by the veins pre-incubation with RuBPY (2 µM) or GTN (4 µM) for 60 min. The eNOS phosphorylated in the activation site (Ser1177) and in the inactivation site (Thr495) was accessed by Western Blotting. The cytosolic concentration of NO ([NO]c) was measured by flow cytometry and confocal microscopy. 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). Both NO donors increased [NO]c in vascular smooth muscle cells. Previous exposure for 10 min with RuBPY (2 µM or 10 µM) or GTN (4 µM or 0.1mM) did not induce tolerance. However, 30 min pre-exposure did not change the relaxation to RuBPY, but it reduced the relaxation to GTN (with 4 µM, ME: 45.4 ± 2.2%, n= 6, P<0.05, and 0.1 mM, ME: 39.2 ± 1.4%, n= 6, P<0.05). Pre-exposure for 60 min with RuBPY reduced the relaxation in the concentrations of 2 µM (ME: 48.0 ± 2.3%, n= 7, P<0.05) and to 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 acetylcholine was induced only with GTN. The ME stimulated with acetylcholine was reduced from100.3 ± 5.3% to 75.1 ± 4.2% (n=7, P<0.05). Whereas RuBPY and GTN had phosphorylated eNOS in Thr495, only GTN phosphorylated eNOS-Ser1177. In conclusion, our data demonstrate that RuBPy induces tolerance in both concentrations, only after 60 min exposure. It does not induce cross-tolerance to acetylcholine and SNP. It does not interfere with the activation site of eNOs.