In this study we describe the characterization of a panel of 12 anti-mouse CD40 monoclonal antibodies (mAb). Characterization was performed in terms of antibody-binding site relative to the CD154 ligand, and the relationship between position and functional outcome of binding. The antibodies divided into three groups. The first were strong inhibitors of CD154 binding, and induced strong proliferative and activation signals to B cells. Two antibodies gave intermediary inhibition and comparable levels of activation. The remaining antibodies were found to bind outside the CD154 binding site and were poor inducers of B-cell activation. Data presented show a strong correlation between location of mAb binding and the resultant activation signal delivered. This correlation is shown to be independent of the isotype of the antibody involved and of its affinity. Implications of these findings are discussed.