Bacterial, but not mammalian DNA, can induce interferon-gamma (IFN-gamma) in murine splenocytes. To elucidate the basis of this activity, we have assessed in vitro cytokine production by C3H/HeJ splenocytes stimulated with either DNA from Escherichia coli or a synthetic oligonucleotide containing an active palindromic sequence identified from DNA. Both DNAs induced IFN-gamma production, with the requirement for intact DNA shown by sensitivity to DNase digestion. Fractionated cell populations were evaluated to determine direct or indirect cellular effects of the DNA. Although bacterial DNA failed to induce IFN-gamma in the nonadherent cell population, supernatants from adherent cells stimulated by DNA induced IFN-gamma production by these cells. Interleukin-12 (IL-12) was detectable in supernatants from DNA-stimulated splenocytes before IFN-gamma, and neutralizing antibodies directed against IL-12 markedly inhibited the induction of IFN-gamma. Anti-tumor necrosis factor-alpha (TNF-alpha) antibodies also inhibited IFN-gamma production, and the combination of both anti-IL-12 and anti-TNF-alpha could totally inhibit production of IFN-gamma. Taken together, these results indicate that the stimulation of IFN-gamma production by bacterial DNA is mediated by IL-12 and TNF-alpha and point to macrophages/monocytes as targets of action of this macromolecule.