Bacterial genomic DNA, plasmid DNA (pDNA) and synthetic oligodeoxynucleotides (ODN) containing immunostimulatory DNA sequences (ISS) have been proposed to foster a T_h1 response via the release of type 1 cytokines from macrophages, dendritic cells, NK cells and B cells. In this study, we show that ISS-enriched DNA up-regulates a distinct profile of cell surface molecules on macrophages and B cells in vitro and in vivo. ISS-ODN and ISS-containing pDNA enhanced the expression of antigen presentation molecules (MHC class I and II), co-stimulatory molecules (B7-1, B7-2 and CD40), cytokine receptors (IFN-γ receptor and IL-2 receptor), an adhesion molecule (ICAM-1) and an Fc receptor (Fcγ receptor) on murine B cells or bone marrow-derived macrophages. The increased expression of these surface molecules is seen in purified cell populations and is largely independent of the effects of type 1 cytokines. Splenic antigen-presenting cells stimulated with ISS-ODN in vivo efficiently activate naive T cells and bias their differentiation toward a T_h1 phenotype in vitro . Thus, the induction of both type 1 cytokines and a distinct profile of cell surface molecules contributes to the potent immunostimulatory effects of ISS-containing DNA on innate and adaptive immunity.