Synthetic oligonucleotides (ODNs) containing immunostimulatory CpG motifs (CpG) are a new class of adjuvants suitable for the development of recombinant vaccines. Here we describe that endogenous interferon (IFN) was critical for the adjuvant activity of CpG ODN as genetically deficient mice developed significantly lower IgG antibody titers following immunization with recombinant proteins. In contrast, the absence of endogenous IL-12/IL-23 or IL-4 had little impact on the magnitude of the antibody response but instead caused a dramatic change in the pattern of IgG isotypes. The dependence on IFN-gamma was specific for CpG ODN and it was not observed with other adjuvants tested. IFN-gamma was produced by NK, dendritic cells, CD4+ and CD8+ T cells stimulated in vitro with CpG ODN. Adoptive transfer experiments confirmed that CD4+ or CD8+ T cells were in fact relevant sources of IFN-gamma in vivo. Following CpG ODN injection, splenic dendritic cells from IFN-gamma deficient mice did not up-regulate CD86 or CD40 expression, suggesting a role for these molecules. The importance of CD28 (CD86 ligand) was confirmed using CD28 deficient mice which presented severely impaired immune responses following CpG ODN-assisted immunization.