Aim:To study the relationship between primary structures of oligodeoxynucleo-tides (ODN) containing unmethylated deoxycytidyl-deoxyguanosine (CpG)dinucleotide motifs and their immunostimulatory activities in mouse spleen cells.Methods:A series of CpG ODN with different primary structures were synthesized.Their capabilities to stimulate mouse spleen cell proliferation were determined by[~3H]thymidine incorporation assay.Cytokine (interleukin [IL]-6,IL-12,and IFN-α)secretion spectra induced by CpG ODN were assessed by ELISA.The ability ofCpG ODN to activate natural killer cells was evaluated by standard 4 h ~(51)Cr-releaseassay.Flow cytometry was utilized to examine the expressions of various lympho-cyte surface molecules on diverse immunocytes.An effective CpG ODN for murine,ODN1826,was set as the template of modification and the positive control.Results:The immunostimulatory activities of CpG ODN with different sequencesand compositions varied markedly,both in character and in extent.It was uselessfor improving the immunostimulatory activity of ODN1826 by simply increasingthe functional hexameric CpG motif number,modifying the site of CpG motifs,orchanging the distance between multi-CpG motifs.However,an addition of a self-complementary palindrome structure at the 3’-end,but not the 5’-end of CpG ODN,aroused marked improvement in its activity.Several designed ODN had superiorcomprehensive immunostimulatory properties compared to ODN 1826.Conclusion:The immunostimulatory activity of a CpG ODN was relevant to its primary structure.It was useless for promoting immunostimulatory activity to simply change CpGmotif number,space,or distance.The 3’-end palindrome structure of CpG ODN isassociated with enhanced immunostimulatory activity. Aim: To study the relationship between primary structures of oligodeoxynucleo-tides (ODN) containing unmethylated deoxycytidyl-deoxyguanosine (CpG) dinucleotide motifs and their immunostimulatory activities in mouse spleen cells. Methods: A series of CpG ODN with different primary structures were synthesized .ir Capabilities to stimulate mouse spleen cell proliferation were determined by [~ 3H] thymidine incorporation assay. Cytokine (interleukin [IL] -6, IL-12, and IFN-α) secretion spectra induced by CpG ODN were assessed by ELISA. ODN to activate natural killer cells was evaluated by standard 4 h 51 Cr-release assay. Flow cytometry was utilized to examine the expressions of various lympho-cyte surface molecules on diverse immunocytes. An effective CpG ODN for murine, ODN 1826, was set as the template of modification and the positive control. Results: The immunostimulatory activities of CpG ODN with different sequences and compositions varied markedly, both in character and in extent. It was useless for improving the immunostimulatory activity of ODN1826 by simply increasing the functional hexameric CpG motif number, modifying the site of CpG motifs, orchanging the distance between multi-CpG motifs.However, an addition of a self-complementary palindrome structure at the 3’- end, but not the 5’-end of CpG ODN, aroused marked improvement in its activity. Selective designed ODN had superior comprehensive immunostimulatory properties compared to ODN 1826. Conlusion: The immunostimulatory activity of a CpG ODN was relevant to its primary structure. It was useless for promoting immunostimulatory activity to simply change CpG motif number, space, or distance. The 3’-end palindrome structure of CpG ODN isassociated with enhanced immunostimulatory activity.