The hemes of hydroxylamine oxidoreductase (HAO) have been analyzed optically by potentiometric titrations using a low volume optically transparent thin layer electrochemical cell. The electrochemical behavior of the HAO monomeric unit has been interpreted by modeling the spectroelectrochemical data at several wavelengths to eight one-electron Nernst sites: seven c-type hemes and one P460 heme. Of the seven c-hemes, six show alpha-bands with absorption maxima at or near 553 nm. One c-heme has an alpha-band absorption maximum at 559 nm. The six c-553 hemes have midpoint potentials at pH 7.0 of +288, -10, -162, -192, -265 and -412 mV versus the normal hydrogen electrode (NHE). The c-559 heme has a midpoint potential (Em') at pH 7.0 of +11 mV versus NHE. The midpoint potential of the P460 heme is at -260 mV versus NHE at pH 7.0. In contrast, the midpoint potential for the P460 heme in another protein, cytochrome P460, from the same organism is -402 mV versus NHE at pH 7.0. Midpoint potentials of the c-hemes show little, if any, pH dependence over the range of pH 6-8. In contrast, Em' for the P460 heme changes with a slope of -60 mV/pH unit over the same range. Electrochemical isolation of the P460 heme at pH 8.0 led to the discovery of a broad spectroscopic feature centered near 740 nm that was assigned to the oxidized P460 heme. Changes in the spectroelectrochemical behavior of HAO after inactivation by H2O2 was almost exclusively restricted to the P460 heme of HAO. Both the 464-nm absorption band of the reduced P460 heme and the 740-nm band of the oxidized heme were no longer present. For the c-hemes, the only effect seems to be a slight shift in Em' for a single c-553 heme from -162 to -135 mV.