Cationic lipid-DNA complexes (CLDC) are reported to be safe and effective for systemic gene delivery, particularly to the lungs. However, we observed that iv injection of CLDC induced immunologic effects not previously reported. We found that even very low doses of CLDC administered iv induced marked systemic immune activation. This response included strong up-regulation of CD69 expression on multiple cell types and systemic release of high levels of Th1 cytokines, from both lung and spleen mononuclear cells. CLDC were much more potent immune activators on a per weight basis than either LPS or poly (I: C). The remarkable potency of CLDC appeared to result from enhancement of the immune stimulatory properties of DNA, since cationic lipids alone were without immune stimulatory activity. Systemic treatment with CLDC controlled tumor growth and significantly prolonged survival times in mice with metastatic pulmonary tumors. NK cells accumulated to high levels in the lungs of CLDC-treated mice, were functionally activated, and released high levels of IFN-γ. The antitumor activity induced by CLDC injection was dependent on both NK cells and IFN-γ. Thus, DNA complexed to cationic liposomes becomes highly immunostimulatory and capable of inducing strong antitumor activity when administered systemically.