Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl‐deoxyguanosine (CpG) dinucleotides (CpG ODN) mimic the immunostimulatory activity of bacterial DNA and are recognized by the Toll‐like receptor 9 (TLR9). CpG ODN of the B‐Class stimulate strong B cell and NK cell activation and cytokine production. The highest degrees of NK stimulation as well as IFN‐α secretion by plasmacytoid DC were found to occur only with A‐Class ODN. A third class of CpG ODN combines the immune effects of A‐ and B‐Class CpG ODN. C‐Class ODN strongly stimulate B cell or NK cell activation and IFN‐α production. In contrast to the A‐Class, the C‐Class is wholly phosphorothioate, has no poly‐G stretches, but has palindromic sequences combined with stimulatory CpG motifs. All classes stimulate TLR9‐dependent signaling, but with strikingly different dose‐response relationships that are quite in contrast to those observed for IFN‐α. Effects similar to those on human cells were observed on mouse splenocytes. In contrast, splenocytes from TLR9‐deficient mice did not show any response to the three CpG ODN classes. In vivo studies demonstrate that C‐Class ODN are very potent Th1adjuvants. C‐Class ODN may represent new therapeutic drugs that combine the effects of A‐ and B‐Class ODN for broad applications in infectious disease or cancer therapy.