During catalysis, the heme in nitric oxide synthase (NOS) binds NO before releasing it to the environment. Oxidation of the NOS ferrous heme-NO complex by O2 is key for catalytic cycling, but the mechanism is unclear. We utilized stopped-flow methods to study the reaction of O2 with ferrous heme-NO complexes of inducible and neuronal NOS enzymes. We found that the reaction does not involve heme-NO dissociation, but instead proceeds by a rapid direct reaction of O2 with the ferrous heme-NO complex. This behavior is novel and may distinguish heme-thiolate enzymes, such as NOS, from related heme proteins.