DNA is a complex macromolecule the immunological properties of which depend on short sequence motifs called CpG motifs or immunostimulatory sequences (ISS). These sequences are mitogenic for B cells and can stimulate macrophage cytokine production. While these sequences do not directly activate T cells, they can augment effects of stimulation via the TCR. Furthermore, ISS can affect T cells because of macrophage production of IL-12 and IFN-alpha/beta. In these studies, we further evaluated the immune effects of DNA on T cells, testing the possibility that certain T cell populations can respond directly to this stimulus. We therefore tested the in vitro responses of thymocytes to a series of phosphodiester (Po) and phosphorothioate (Ps) oligonucleotides (ODNs) varying in sequence. In in vitro cultures, phosphorothioate ODNs (sODNs) containing CpG motifs induced significant proliferation of murine thymocytes, although phosphodiester compounds lacked activity. The magnitude of stimulation varied with sequences flanking the CpG motifs, as both dA and dT sequences enhanced the stimulatory capacity of the CpG motif. Furthermore, CpG sODNs were strong costimulators of anti-CD3-mediated thymocyte activation, increasing proliferation compared to anti-CD3 in the absence of DNA. This activation was only partially inhibited by cyclosporine A and was not dependent on a calcium influx. Together, these results indicate that phosphorothioate oligonucleotides containing CpG motifs can directly induce thymocyte proliferation as well as augment TCR activation. These observations thus extend the range of actions of CpG DNA and suggest additional mechanisms for its function as an immunomodulatory agent or adjuvant.