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To sort out the putative roles of endogenous hydrogen sulfide (H2S) in clinical conditions wherein systemic inflammation or hypoxia is present, it becomes crucial to develop approaches capable of affecting H2S concentration that can be safely applied in humans. We have investigated a paradigm, which could achieve such a goal, using vitamin B12 (vit.B12), at the dose recommended in cyanide poisoning, and very low levels of methemoglobin (MetHb). Hydroxocobalamin in the plasma, supernatant of kidney, and heart tissue homogenates of rats that had received vit.B12 (140 mg.kg(-1) intravenous) was found in the μM range. Exogenous H2S (100 μM) added to the plasma or supernatants of these rats decreased at a significantly higher rate than in control rats. In the latter however a spontaneous oxidation of exogenous H2S occurred. In vitro, hydroxocobalamin solution (100 μM) decreased, within <2 min, an equimolar concentration of H2S by 80%. Three to five percent MetHb prevented H2S induced hyperventilation in vivo and decreased exogenous H2S in vitro by 25-40 μM within 30 s. Our observations lead to the hypothesis that innocuous levels of MetHb and vit.B12 could be a used as an effective and safe way to test the role of endogenous H2S in vivo.