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The vertebrate immune system recognizes bacterial DNA based on the presence of unmethylated CpG-dinucleotides in particular base contexts (“CpG motifs”). In contrast to mice, knowledge about CpG-mediated effects on human B cells is poor. In the present study we identify and determine an optimal human CpG motif. A phosphodiester oligonucleotide containing this motif strongly stimulated CD86, CD40, CD54, and MHC class II expression, IL-6 synthesis, and proliferation of primary human B cells. These effects required internalization of the oligonucleotide and endosomal maturation. The molecular mechanism of action of this CpG motif was associated with the sustained induction of the NF-κB p50/p65 heterodimer and of the transcription-factor complex AP-1. Transcription-factor activation by CpG DNA was preceded by increased phosphorylation of the stress kinases c-Jun N-terminal kinase and p38, and of …