Cystathionine β-synthase (CBS) is a pyridoxal phosphate-dependent enzyme that catalyzes the condensation of homocysteine with serine or with cysteine to form cystathionine and either water or hydrogen sulfide, respectively. Human CBS possesses a noncatalytic heme cofactor with cysteine and histidine as ligands, which in its oxidized state is relatively unreactive. Ferric CBS (Fe(III)-CBS) can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NO·), leading to inactive enzyme. Alternatively, Fe(II)-CBS can be reoxidized by O2 to Fe(III)-CBS, forming superoxide radical anion (O2 -·). In this study, we describe the kinetics of nitrite (NO2·-) reduction by Fe(II)-CBS to form Fe(II)NO·-CBS. The second order rate constant for the reaction of Fe(II)-CBS with nitrite was obtained at low dithionite concentrations. Reoxidation of Fe(II)NO·-CBS by O2 showed complex kinetic behavior and led to peroxynitrite (ONOO-) formation, which was detected using the fluorescent probe, coumarin boronic acid. Thus, in addition to being a potential source of superoxide radical, CBS constitutes a previously unrecognized source of NO· and peroxynitrite.