Cholera toxin (CT) is a potent mucosal immunogen and adjuvant that can strongly prime mucosal T cells. The present study was undertaken to investigate the effects of CT on the expression and functional activity of the costimulatory molecules B7.1 and B7.2 on macrophages and the relationship of these effects to the mucosal adjuvanticity of CT. Bone marrow macrophages (BMM) were generated by culturing bone marrow with macrophage CSF or granulocyte-macrophage CSF. After treatment with either CT alone or IFN-gamma alone, B7.2 expression on BMM was moderately up-regulated and was further increased when BMM were treated with both CT and IFN-gamma together. Interestingly, CT had no effect on B7.1 expression despite the close relationship between these two molecules. 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 functional costimulatory activity of macrophages for both anti-CD3-stimulated and allostimulated T cells, an increase that was blocked by anti-B7.2, but not anti-B7.1, Ab. B7.2 expression by Mac1+ Peyer's patch cells was increased after intraluminal exposure to CT in vivo. Treatment of mice with anti-B7.2 Ab in vivo inhibited both the mucosal adjuvanticity and the immunogenicity of CT. We conclude that CT enhances the costimulatory activity of mucosal APC by differentially up-regulating B7.2 expression, an effect that appears to be important for its mucosal adjuvanticity and immunogenicity.