The role of reactive nitrogen intermediates (RNI) such as nitric oxide (.NO) in host defense against pyogenic microorganisms is unclear, and the actual interactive effect of RNI and reactive oxidative intermediates (ROI) for microbial killing has not been determined. Since, in nature, ROI and RNI might be generated together within any local infection, we evaluated the separate and interactive effects of .NO and O2- on staphylococcal survival by using a simplified system devoid of eukaryotic cells. These studies showed that prolonged exposure of staphylococci to .NO does not result in early loss of viability but instead is associated with a dose-related delayed loss of viability. This effect is abrogated by the presence of hemoglobin, providing further evidence that the effect is RNI associated. Superoxide-mediated killing also is dose related, but in contrast to RNI-mediated killing, it is rapid and occurs within 2 h of exposure. We further show that the interaction of .NO and O(2)- results in decreased O(2)--mediated staphylococcal killing at early time points. .NO, however, appears to enhance or stabilize microbial killing over prolonged periods of incubation. This study did not produce evidence of early synergism of ROI and RNI, but it does suggest that .NO may contribute to host defense, especially when ROI-mediated killing is compromised.