CD95 (Fas/APO-1)-mediated apoptosis appears to be regulated by positive and negative signaling molecules. A human CD40/CD95 chimeric receptor was stably transfected into CD95-expressing human Jurkat T cells, and signaling through native and chimeric CD95 was compared in the same cell type to assess contributions of the CD95 extracellular and intracellular domains. Apoptosis was induced in these transfectants by soluble CD40 ligand and also by the anti-CD40 monoclonal antibodies (mAb) M2 and M3. The M2 mAb was more effective than M3 in these transfectants. In contrast to apoptosis mediated through native CD95, CD40/CD95-mediated apoptosis was not inhibited by phorbol-12-myristate-13-acetate (PMA). The apoptotic response to the anti-CD40 mAb M3, but not M2, was enhanced by PMA and dibutyryl cyclic adenosine monophosphate (db-cAMP), which also increased mRNA levels and surface expression of CD40/CD95. The enhancing effects of PMA, but not those of db-cAMP, were sensitive to cycloheximide. The M2 and M3 mAbs appeared to have virtually identical binding affinities but, when added to cells together, M3 inhibited M2-induced apoptosis. These mAbs may bind neighboring epitopes, but M2 induces a more effective signaling-competent conformation upon the chimeric receptor. These data suggest that dimerization, however only in a signaling-competent conformation, was sufficient to induce apoptosis. When expressed as a chimera with the CD40 extracellular domain, the CD95 intracellular domain was not inhibited by protein kinase C (PKC)-dependent pathways, suggesting that the CD95 extracellular domain is required for association with a molecule that inhibits the apoptotic signal.