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It is known that cyanide is converted to thiocyanate in the presence of the enzyme rhodanese. The enzyme is activated by sulfur transfer from an appropriate sulfur donor. The activated enzyme then binds cyanide and transfers the sulfur atom to cyanide to form thiocyanate. This project began as an exploration of the ability of disulfides to act as sulfur donors in the rhodanese-mediated detoxification of cyanide. To our surprise, and contrary to expectations based on efficacy studies in vivo, our in vitro results showed that disulfides are rather poor sulfur donors. The transfer of a sulfur atom from a disulfide to the enzyme must occur via cleavage of a carbon-sulfur bond either of the original disulfide or in a mixed disulfide arising from the reaction of rhodanese with the original disulfide. Extending the reaction time and addition of chloride anion (a nucleophile) did not significantly change the results of the experiment. Using ultrasound as a means of accelerating bond cleavage also had a minimal effect. Those results ruled out cleavage of the carbon-sulfur bond in the original disulfide but did not preclude formation of a mixed disulfide. S-Methyl methylthiosulfonate (MTSO) was used to determine whether a mixed disulfide, if formed, would result in transfer of a sulfur atom to rhodanese. While no thiocyanate was formed in the reaction between cyanide and rhodanese exposed to MTSO, NMR analysis revealed that MTSO reacted directly with cyanide anion to form methyl thiocyanate. This result reveals the body's possible use of oxidized disulfides as a first line of defense against cyanide intoxication. The oxidation of disulfides to the corresponding thiosulfinate or thiosulfonate will result in facilitating their reaction with other nucleophiles. The reaction of an oxidized disulfide with a sulfur nucleophile from glutathione could be a plausible origin for the cyanide metabolite 2-aminothiazoline-4-carboxylic acid.