We have recently reported that oligodeoxynucleotides (ODN) that contain a CpG dinucleotide flanked by two purines on the 5'-side and two pyrimidines on the 3'-side induce potent B cell proliferation and differentiation. The present study investigates the role of the ODN backbone in determining the magnitude of the lymphocyte stimulation. Phosphorothioate ODN were approximately 2 logs more potent than the same sequence with a phosphodiester backbone. Chimeric ODN in which the 5'- and 3'-ends were modified with nuclease-resistant internucleotide linkages induced widely varying degrees of immune activation depending on the modification. Phosphorodithioate linkages were by far the most potent and induced B cell activation at nanomolar concentrations, approximately 1 log lower than required for the next most potent modification, phosphorothioate. Methylphosphorothioate terminal linkages were slightly more potent than phosphodiester, which were in turn slightly more potent than terminal methylphosphonate-modified ODN.