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Nitric oxide (NO) plays an important role in the control of the vascular tone, and several reports have demonstrated the relaxing effects of compounds with the functional group C=NOH (oxime) and the participation of NO in these responses. Accordingly, new drugs with potential ability to generate nitric oxide have been studied. The aim of this study was to investigate the mechanisms underlying the vasorelaxant effect induced by D-arabinose oxime (DAO). In phenylephrine-pre-contracted mesenteric rings in the presence or absence of functional endothelium, DAO caused a concentration-dependent relaxation. The vasorelaxant effect induced by this oxime was not modified after pretreatment with L-NAME as compared to control. However, in endothelium-denuded rings, ODQ, hydroxocobalamin and PTIO caused a significant reduction in vasorelaxation induced by DAO when compared to controls. Beyond these functional experiments, a direct measure of NO was carried out by an amperometric technique. In the absence or presence of phenylephrine-pre-contracted artery rings, DAO induced a significant increase in current (nA) that was recorded by the NO sensor, and this increase was attenuated after hydroxocobalamin addition. In conclusion, vasorelaxation induced by DAO is due to nitric oxide release, at least partly spontaneous, and to the activation of the NO/sGC/PKG pathway