During T-APC interactions in vivo, interfering with CD40-CD154 interactions leads to reduced T cell priming, defects in effector function, and, in some cases, T cell tolerance. As shown here, however, presentation of conventional peptide Ags by CD40-deficient spleen APC in vitro leads to normal CD4+ T cell proliferative responses. By contrast, responses to the same peptides presented by purified B cells were markedly reduced in the absence of CD40. Thus, the requirement for CD40-CD154 interactions appears to be strongly influenced by the type of APC involved. Analysis of responses to endogenous superantigens, which are known to be strongly dependent on B cells for presentation, indicated that CD4+ responses to strong Ags are less dependent on CD40 than are responses to weak Ags. Similar findings applied to negative selection in the thymus. Thus, deletion of potentially autoreactive cells depended on CD40 expression when B APC were involved, and this requirement was most pronounced when negative selection was directed to weak Ags.