Nitric oxide (NO) released by activated alveolar macrophages (AM) can mediate effects on target cells and can also react with superoxide anion (O 2-) to form peroxynitrite (PN), a highly cytotoxic product. The role of NO and PN in AM cytotoxicity for normal lung cells was investigated using co-cultures of rat lung fibroblasts (FB) and rat AM treated with lipopolysaccharide+interferon-γ (LI). AM and FB, alone and in co-culture, were treated with LI for 5 days and cell viability measured. The culture media was analyzed for NO, TNF-α, O 2-, and IL-1β. A decreased FB viability was correlated with increased NO release by LI-activated AM. Pretreatment of co-cultures with the inducible NOS inhibitor l-NAME caused dose-related decreases in NO release by AM and increases in FB viability. Although TNF-α release was increased in co-cultures treated with LI, the viability of FB was not affected when cultures were treated with similar concentrations of TNF-α in the absence of AM. O 2- could not be detected in the media and addition of superoxide dismutase (SOD) did not protect FB. These data suggest that neither O 2- nor PN contributed to the loss of cell viability. Activated AM may kill normal rat lung FB through a NO-mediated pathway that does not involve PN.