S-nitrosothiols (RSNO) are considered as potential drugs for delivering nitric oxide (•NO) or related species in cardiovascular disorders associated with decrease in ·NO bioavailability. We have synthesized a new RSNO, i.e. S,S'-dinitrosobucillamine (BUC(NO)2), which combines in its structure two S-mononitrosothiols, S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO). Synthesized BUC(NO)2 was structurally characterized using high-performance liquid chromatography/mass spectrometry (HPLC/MS), 1H nuclear magnetic resonance (1H NMR), infrared (IR) and UV–visible spectroscopies, and thermal analysis; resulting data are consistent with the expected structure. The vasorelaxant effect of BUC(NO)2 was evaluated using isolated rat aortic rings and compared to SNAP, NACNO, and to an equimolar mixture of NACNO plus SNAP in order to mimic the number of •NO contained in a BUC(NO)2 molecule. BUC(NO)2 (pD2=7.8±0.1) was more potent in vasorelaxation than NACNO (pD2=6.4±0.2), SNAP (pD2=6.7±0.1) and the mixture of SNAP plus NACNO (pD2=6.7±0.2). The release of •NO from BUC(NO)2 was 6-fold that of the basal value and significantly higher than the release of •NO from the SNAP plus NACNO mixture (4-fold increase versus basal value). Finally, the role of protein disulfide isomerase (PDI) in BUC(NO)2 metabolism was investigated. Vasorelaxant effect (pD2=6.8±0.2) and •NO release decreased in the presence of a PDI inhibitor (both P<0.05 versus BUC(NO)2). In conclusion, BUC(NO)2 releases a larger amount of •NO into the aorta, partially through PDI activation, and induces vasorelaxation at lower concentrations than other RSNO previously reported.