Both the binding of CpG-oligodeoxynucleotides (CpG-ODNs) to cell-surface and its immunostimulatory activity were modulated by extracellular pH in present study. At neutral pH (pH 7.4), the binding of CpG-ODN to splenocyte-surface, as well as that of non-CpG-ODN, was competitively inhibited by non-specific DNA-Herring sperm DNA in a dose dependent manner, indicating their binding sites have no specificity for CpG-motif. When the extracellular pH shifted to acidic (pH 6.4), however, their binding to cell-surface markedly increased, and only the binding of non-CpG-ODN instead of CpG-ODN was inhibited by Herring sperm DNA, implying such pH change enabled CpG-ODN bind to its specific binding-site. Consistently, lymphocytes appeared more sensitive to the stimulation of CpG-ODN at acidic pH, and Herring sperm DNA inhibited the CpG-ODN-induced TNF production from splenocytes at pH 7.4, but not at pH 6.4. These results suggest the existence of membrane receptor that specifically engages CpG-ODN with high affinity only at acidic pH, and support the hypothesis that the binding CpG-ODN to its specific membrane receptor and subsequently triggering of CpG-related signaling occurred within acidified endosomes.