The reactions of several horse heart myoglobin species with nitrosyl hydride, HNO, derived from Angeli's salt (AS) and Piloty's acid (PA) have been followed by UV–visible, 1 H NMR and EPR spectroscopies. Spectral analysis of myoglobin-derived speciation during the reactions was obtained by using singular value decomposition methods combined with a global analysis to obtain the rate constants of complex sequential reactions. The analysis also provided spectra for the derived absorbers, which allowed self-consistent calibration to the spectra of known myoglobin species. Using this method, the determined rate for trapping of HNO by metmyoglobin, which produces NO-myoglobin, is found to be 2.7×10 5 M −1 s −1 at pH7.0 and 1.1×10 5 M −1 s −1 at pH9.4. The reaction of deoxymyoglobin with HNO generates the adduct HNO-myoglobin directly, but is followed by a secondary reaction of that product with HNO yielding NO-myoglobin; the determined bimolecular rate constants for these reactions are 3.7×10 5 M −1 s −1 and 1.67×10 4 M −1 s −1 respectively, and are independent of pH. The derived spectrum for HNO-myoglobin is characterized by a Soret absorbance maximum at 423nm with an extinction coefficient of 1.66×10 5 M −1 cm −1 . The rate constant for unimolecular loss of HNO from HNO-myoglobin was determined by competitive trapping with CO at 8.9×10 −5 s −1 , which gives the thermodynamic binding affinity of HNO to deoxymyoglobin as 4.2×10 9 M −1 . These results suggest that the formation of HNO-ferrous heme protein adducts represents an important consideration in the biological action of HNO-releasing drugs.