We previously reported that the anti-epidermal growth factor (EGF) receptor monoclonal antibody (mAb) 225 induces DiFi colon cancer cells to undergo apoptosis, and this apoptosis was accompanied by activation of the two apoptosis initiation caspases, caspase-8 and caspase-9. In the current study, we found that pretreatment of DiFi cells with the caspase-8-specific inhibitor z-IETD-fmk but not pretreatment with the caspase-9-specific inhibitor z-LEHD-fmk inhibited mAb 225-induced apoptosis, indicating that caspase-8 plays an essential role in initiating mAb 225-induced apoptosis. Because caspase-8 is activated primarily by the members of the tumor necrosis factor (TNF) receptor family, such as Fas, TNF receptor-1 (TNFR1), or receptors for TNF-related apoptosis-inducing ligand (TRAIL), we investigated whether mAb 225 activated caspase-8 by regulating one or more of these known pathways. Exposure of DiFi cells to TNFalpha or TRAIL activated caspase-8 and induced apoptosis in the cells. A TNFR1-antagonistic mAb or a TRAIL decoy receptor inhibited the activation of caspase-8 and the subsequent apoptosis induced by TNFalpha or TRAIL, respectively, in the cells. However, neither the TNFR1-antagonistic mAb nor the TRAIL decoy receptor inhibited mAb 225-induced activation of caspase-8 and apoptosis in DiFi cells. DiFi cells express detectable level of Fas but are not sensitive to the treatment by the Fas-agonistic mAb CH-11. A Fas-antagonistic mAb (ZB-4) inhibited the Fas-agonistic mAb CH-11-induced caspase-8 activation and apoptosis in Jurkat T-leukemic cells (used as positive control), but had no effect on mAb 225-induced activation of caspase-8 and apoptosis in DiFi cells. Taken together, our results suggest that mAb 225 does not interact with or regulate these known death receptor pathways. An exploration is therefore warranted for a novel mechanism by which mAb 225 activates caspase-8 and triggers apoptosis in DiFi cells.