Detailed kinetic and mechanistic studies have been carried out on the reaction between aquacobalamin/hydroxocobalamin (CbIOH2+/CbIOH) and nitroxyl (HNO) generated by Piloty's acid (PA, N-hydroxybenzenesulfonamide) over a wide pH range (3.5-13). The resulting data showed that in a basic solution HNO can react with hydroxocobalamin to form nitrosylcobalamin despite the inert nature of CbIOH. It was shown that at low PA concentrations the -step is the decomposition of PhSO2NHO- to release HNO, whereas the reaction between CbIOH and HNO becomes the rate-determining step at high PA concentrations. Data from kinetic studies on the reaction of CbIOH with an excess of HNO enabled us to experimentally determine the pK(a)(HNO) value from initial rate data as a function of pH, giving pK(a)(HNO) = 11.47 +/- 0.04. An especially interesting observation was made in the neutral pH range, where PA is stable and does not produce HNO. Under such conditions, rapid formation of CbINO was observed in the studied system. The obtained data suggest that CbIOH2+ reacts directly with PA to form a Piloty's acid-bound cobalamin intermediate, which deprotonates rapidly at neutral pH followed by rate-determining S-N bond cleavage to give CbINO and release PhSO2-.