A co-culture cytotoxicity model was developed to investigate the interaction between activated alveolar macrophages (AM) and other lung cells. Freshly lavaged rat AM were co-cultured with normal rat lung fibroblasts (RLFB) at a 5:1 ratio of AM: RLFB. Co-cultures were treated with bacterial endotoxin + interferon (LI) overnight to activate AMs. RLFB viability was significantly decreased (80%) in LI-treated co-cultures by day 5. AM had no effect on RLFB viability in the absence of LI. LI caused a slight decrease (15%) in RLFB viability in the absence of AM. This co-culture model was used to investigate the role of peroxynitrite in the toxicity of activated AM for RLFB. LI stimulated an increased release of nitric oxide (NO) by AM. LI-activated AM were stimulated to release a significant amount of NO into the medium. Pretreatment of AMs with L-NAME, an inhibitor of inducible NO synthetase, protected RLFB, indicating the NO was a key toxic component. In addition to NO, activated AM have been reported to release superoxide anion (O2-). These two intermediates can combine to form peroxynitrite (PN) a reactive and toxic intermediate. LI stimulated an increased release of NO by AM; however, O2- could not be detected in the media from co-cultures, and the addition of superoxide dismutase did not protect RLFBs. As a further check for the presence of PN, RLFB were immuno-stained for N-tyrosine, a marker for PN reaction. However, there was no apparent staining of cells for N-tyrosine. These findings indicate that NO toxicity for normal lung cells is not mediated by PN. LI also stimulated an increased release of tumor necrosis factor-alpha (TNFa) from AM. However, treatment of RLFB with the same concentrations of TNFa detected in the co-culture media had no effect on RLFB viability. These data suggest that AM cytotoxicity for RLFB is mediated by NO and not TNFa. IL-1 was not detected in the medium before or after LI-activation.