Plasmid DNA (pDNA) is very important in non-viral gene therapy and DNA vaccination. Unmethylated CpG motifs in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response, which inhibits gene expression while improving immunological consequences. In this report, we investigated the cytokine secretion induced by pDNA/cationic liposome complexes using murine macrophages. Naked CpG DNA induced tumor necrosis factor-alpha (TNF-alpha) secretion from the macrophages. but DNA without CpG motif did not, demonstrating that the cytokine induction was mediated by CpG motifs. pDNA complexed with cationic liposomes, but not the cationic liposomes alone, produced a significant amount of TNF-alpha from the macrophages. Surprisingly, methylated pDNA and calf thymus DNA complexed with the cationic liposomes were also able to induce TNF-alpha production, indicating that these responses were not dependent on CpG motifs. Taken together, the present study demonstrated that for the first time DNA can stimulate murine macrophages in a CpG motif-independent manner when it is complexed with the cationic liposomes. (C) 2002 Elsevier Science (USA). All rights reserved.