We have shown that surfactant protein A (SP-A) mediates alveolar macrophage (AM) killing of Mycoplasma pulmonis. We have now performed a series of experiments to identify the nature of the reactive species involved. AMs from mycoplasma resistant C57BL/6 mice were activated by incubation with 100 U/ml IFN-γ for 18h, further incubated with NG-monometoyl-L-arginine (NMMA) (1mM), superoxide dismutase (SOD) (100U/ml), or SOD plus catalase (500or 10,000U/ml) for 30 min. SP-A (25 μg/ml) was then added to the AM containing medium for 30 min. AMs were washed to remove unbound SP-A and infected with 1010 colony forming units of M. pulmonis. Maximal killing of mycoplasmes occurred in the presence of SP-A within 6 hours postinfection. NMMA or SOD alone abrogated SP-A mediated mycoplasmal killing while catalase had no effect. The peroxynitrite generator SIN-1 (1mM) incubated with log phase M pulmonis at 37°C in the absence of AMs resulted in significant killing of mycoplasmes by 45 min and total killing by 90 min. These data indicate that AM killing of mycoplasmes is mediated by peroxynitrite, formed by the reaction of nitric oxide with superoxide.