Many of the biologic activities of phosphorothioate oligodeoxynucleotides (PS-oligos) are affected by the sense of chirality of the phosphorus atoms of the internucleotide linkages. Some of the activities are increased by the Rp stereoisomer, and others are increased by the Sp stereoisomer. In previous studies, we showed that PS-oligos containing unmethylated CpG dinucleotides in particular sequence contexts can stimulate B cells and other immune cells. These CpG PS-oligos trigger mitogenactivated protein kinase (MAPK) signaling pathways, causing the induction of B cell proliferation and cytokine and immunoglobulin secretion. We investigated whether the immune stimulation by CpG PS-oligos depends on the sense of their P-chirality. CpG PS-oligos synthesized with internucleotide phosphorothioates of Rp configuration at P-atom showed much stronger MAPK activation and induction of I kappa B degradation after 40 minutes of stimulation compared with PS-oligos synthesized with Sp linkages. In order to determine if the enhanced stimulatory effects of the Rp stereoisomer may result from differential cellular uptake, we examined the rates at which fluorescently labeled Rp or Sp CpG PS-oligos were taken up by B cells, but these were found to be identical to each other and to stereorandom PS-oligos. The stronger stimulatory effect of the R stereoisomer did not last for 48 hours, and (3)H-thymidine incorporation assays at this point showed that only the S stereoisomer was active--to approximately the same level as induced by PS-oligos with stereorandom phosphorothioate linkages. This loss of activity of the R stereoisomer most likely resulted from rapid degradation of the oligonucleotides rather than from reduced interaction with the CpG receptor because PS-oligos in which only the CpG dinucleotide was stereodefined were most stimulatory when the CpG was Rp but not when the CpG was Sp. These studies demonstrate that the sense of Pchirality of PS-oligos plays a major role in determining the biologic activities of CpG motifs. Rp-chirality at the CpG is preferred for best stimulation at early time points, but Sp-chirality of the PS-oligo appears to improve stability and may provide more durable effects in prolonged tissue culture systems.