DNA containing unmethylated CpG dinucleotides (CpG DNA) is a potent activator of innate and acquired immune responses. Although the sequence-specific immunostimulatory activity of CpG DNA has been extensively explored, little information is available about the importance of the stereochemical properties of CpG DNA. In this study, Y-shaped oligodeoxynucleotides (Y-ODNs) were prepared using three ODNs with the halves of each ODN being partially complementary to a half of the other two ODNs. Y-ODN induced greater amounts of tumour necrosis factor-α and interleukin-6 from RAW264.7 macrophage-like cells than did conventional single-stranded ODN (ssODN) or double-stranded ODN (dsODN). The Y-ODN was less stable in serum than dsODN, but greater amounts of Y-ODN were taken up by macrophage-like cells compared with dsODN. A newly designed Y-ODN containing three potent CpG motifs generated significantly higher levels of cytokines compared with dsODN containing the identical sequences. These results indicate that the Y-shaped form of ODN is a novel, reproducible and reliable approach to enhancing the immunostimulatory activity of ODNs.