We generated and characterized novel antibodies specific for a cleavage site of human caspase-8/FLICE and its substrate, FLICE-like inhibitory protein (FLIP). The synthetic peptides used as immunogens were CQGDNYQKGIPVETD (#791) and VSEGQLEDSS-LLEVD (#1342), which corresponded to cleaved regions of N-terminal fragments of caspase-8 and FLIP generated by active caspase-8, respectively. Each antibody purified from rabbit antiserum reacted specifically with the immunogen but not with the peptide corresponding to the unproteolyzed form, as assessed by ELISA. In vitro cleavage of GST-FLIP by active caspase-8 generated an N-terminal fragment (GST-p 43) and a C-terminal one (p 12). Consistent with other in vivo data, the FLIP cleavage site follows the Asp residue, LEVD₃₇₆ GPAMKNVEF, identified on N-terminal sequencing of the p 12 fragment. #1342-antibody (#1342-Ab) recognized the GST-p 43 fragment but not the uncleaved protein, thus confirming its specificity. When the antibodies were used for immunoblot-ting, flow cytometry, and confocal laser microscopy, the proteolysis of caspase-8 and FLIP, and the subcellular localization of their digests could be monitored in apoptotic U 937 cells. Interestingly, a significant increase in the percentage of cells exhibiting caspase-8 and FLIP cleavage was observed upon Fas stimulation in interferon-γ-treated U 937 cells, in which the susceptibility to Fas is extremely enhanced. In contrast, U 937 cells treated with vitamin D₃ or all-trans retinoic acid showed Fas-resistance, and caspase-8 processing and FLIP cleavage were strongly inhibited. In conclusion, we established a system based on the cleavage site-directed antibodies to monitor the dynamics of caspase-8 processing and activation during apoptosis. Using this system, we found that Fas-susceptibility changes during U 937 differentiation occur upstream of caspase-8 processing/activation.