Since Fas-induced apoptosis is major pathway to eliminate unwanted or uncontrolled cells, many types of human cancer cells develop tactful mechanisms to get resistance against the apoptosis. One of the resistant mechanisms in human cancer is overexpression of FLICE-inhibitory protein,(c-FLIP), human homolog of viral protein v-FLIP. c-FLIP has multiple splice variants at transcriptional level or two isoforms at protein level, a long (c-FLIPL) and a short form of c-FLIP (c-FLIPS). However, functional differences between these variants are not fully understood. In this study, we show that c-FLIPL but not c-FLIPS physically binds to Daxx through interaction between C-terminal domain of c-FLIPL and Fas-binding domain of Daxx, an alternative Fas signaling adaptor. Fas-induced cell death and JNK activation are sensitive to Fas stimulation in cell lines carrying undetectable level of c-FLIPL. To support this, overexpression of c-FLIPL but not of c-FLIPS renders the cells resistant to Fas-induced cell death and to JNK activation. In signaling context, the interaction of c-FLIPL with Daxx is likely to inhibit JNK activation by preventing the normal interaction of Daxx and Fas, which is known to lead to apoptosis via JNK activation. This study implies that through this new mechanism, c-FLIPL, acting at both FADD- and Daxx-mediated signaling pathways, may be involved in complete inhibition of Fas-induced cell death and may provide an answer to why c-FLIPL is more abundant and effective than c-FLIPS. (C) 2003 Elsevier Inc. All rights reserved.