Many of the effects of CT as a mucosal adjuvant appear to relate to enhanced T cell priming. The purpose of current study was to investigate the effects of CT on B7.1 and B7.2 expression and resulting alteration of the costimulatory activity of macrophages. Bone marrow macrophages (BMW) were generated by culturing bone marrow with M-CSF. After treatment with CT alone or IFM? alone, 87.2 expression on M-CSF generated BMM was moderately up-regulated. However, B7.2 expression was greatly up-regulated when BMM was treated with both CT and IFNy. In contrast, CT had no effect on B7.1 expression. Up-regulation of B7.2 expression by CT was mediated by intracellular cAMP production in that CT-B subunit had no effect and dibutyryl-cAMP could mimic the effect. CT increased costimulatory activity of macrophages in both anti-CD3 stimulation and allostimulation of T cells, an increase which was blocked by anti-B7.2, but not anti-B7.1 antibody. The variation in the costimulatory capacity of the macrophage preparations correlated well with their level of B7.2 expression. We conclude that CT enhances costimulatory activity of macrophages by selectively up-regulating B7.2 expression, an effect that may be central to its mucosal immunogenicity and adjuvanticity.