Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to trigger apoptosis in many malignant cells. Whereas cancer cells are responsive to TRAIL-induced cell death when used alone or in combination with other agents, normal cells are known to be relatively less sensitive to the ligand, making it a desirable therapeutic compound to target a variety of cancers. TRAIL induces apoptosis through its interaction with its two proapoptotic death receptors (DRs), DR4 and DR5. In addition, it may also bind the decoy receptors (DcRs), DcR1 and DcR2, which lack an intracellular signaling domain, thus negatively regulating TRAIL-induced apoptosis. Previously, it has been shown that interleukin (IL)-8 is elevated in the ascites of patients with ovarian cancer. Therefore, we examined the role that IL-8 may play in modulating sensitivity to TRAIL-mediated apoptosis. We treated the TRAIL-sensitive cell line OVCAR3 with TRAIL over a period of time with or without pretreatment with IL-8. Here we show the novel findings that IL-8 blocks TRAIL-induced cell death and was able to turn the TRAIL-sensitive cell line into a TRAIL-resistant one. We hypothesized that decreased expression of DRs DR4 and DR5 may contribute to TRAIL resistance. Both reverse transcription-PCR and flow cytometry revealed a decrease in DR4 expression after pretreatment of OVCAR3 cells with IL-8. We have also shown that TRAIL was able to induce caspase-8 cleavage in these cells, whereas pretreatment with IL-8 blocked this caspase cleavage. Through array analysis and confirmation with other techniques, we have determined that IL-8 regulates the expression of a member of the mitogen-activated protein kinase superfamily, p38gamma. These findings provide important insights into the modulation of apoptosis by TRAIL and IL-8 in ovarian cancer. The data suggest a potentially important role of IL-8 in protecting ovarian cancer cells from TRAIL-mediated apoptosis and signify a new potential chemotherapeutic target to augment TRAIL therapy.