Unmethylated CpG dinucleotides in DNA contribute to a rapid inflammatory response in mammals. Here we show that N-6-methyladenine (N-6-MeA), a bacterium-specific modified base, also causes cytokine production. An oligodeoxyribonucleotide (ODN) containing N-6-MeA induced cytokines when injected into mice. Co-injection of N-6-MeA and CpG ODNs enhanced cytokines 2- to 3-fold, as compared with the injection of a CpG ODN alone. Plasmid DNA containing N-6-MeA, complexed with cationic lipids, induced IL-12. These results indicate that the bacterium-specific base, in addition to the unmethylated CpG motif, triggers the mammalian immune response, and suggest that N-6-MeA-containing DNA could be useful for cellular immunotherapy and DNA vaccine. (C) 2004 Elsevier Inc. All rights reserved.