Recombinant plasmid DNA alone or in conjunction with a gene delivery system has been used increasingly for in vivo gene transfer. However, little is known about the direct biological effects of plasmid DNA. In this study, we demonstrated that tracheal administration of a number of plasmid DNA protected rats against oxygen toxicity. This protection required the presence of intact plasmid DNA, was not due to endotoxin contamination, and was not shared by salmon testis DNA. The plasmid DNA-induced protection against oxygen toxicity was associated with the production of tumor necrosis factor (TNF) and the enhancement of pulmonary Mn-superoxide dismutase (MnSOD), CuZnSOD, and glutathione peroxidase activities. Coadministration of plasmid DNA and anti-TNF antibody (but not nonspecific IgG) partially abolished the protective effect and reduced the pulmonary MnSOD activity, suggesting that the plasmid DNA-induced oxygen tolerance was in part mediated by the endogenous TNF and MnSOD. In view of these observations and the known immunostimulatory effects of bacterial DNA, caution should be exercised in interpreting the results of in vivo gene transfer using recombinant plasmid DNA.