Heptylprodigiosin (HPDG) is a cytotoxic tripyrole metabolite isolated from a marine bacterium Pseudovibrio denitrificans strain Z143-1. In this study, we investigated the mechanism by which HPDG induces cytotoxicity in human leukemia Jurkat T cells. HPDG-induced apoptosis (classic apoptosis) was shown by G0/G1 arrest and DNA fragmentation that is concentration- and time-dependent in CD95 (Fas/Apo-1)- sensitive Jurkat T cells (S-Jurkat). The MTT cytotoxicity assay is consistent with the apoptosis assay showing an IC50 of ~2 ÁM in S-Jurkat T cells. HPDG-mediated apoptosis in S-Jurkat T cells is dependent on caspase activation based on a caspase 3-like activity assay and the time-dependent immunoblot analysis of caspases. Flow cytometry analysis of CD95-deficient JurkatR, Jurkat/FADD-/-, Jurkat/caspase 8-/-, and vector control cells (Jurkat/A3), showed that HPDG-induced apoptosis requires the CD95 receptor, FADD, and caspase 8. Immunoblot analysis and a mitochondrial dissipation assay revealed delayed activation of the intrinsic pathway. HPDG-treated Jurkat/Neo, Jurkat/Bcl-2+/+ and Jurkat/Bcl-xL+/+ cells showed that HPDG-induced apoptosis is independent of Bcl-2 and Bcl-xL overexpression, suggesting that HPDG is unaffected by the protective mechanism of these anti-apoptotic proteins in Jurkat T cells. This is the first report of the apoptotic mechanism of HPDG that may have clinical utility as a potent chemosensitizer to tumor cells with CD95 expression, dysfunctional apoptosome activity, and overexpressed Bcl-2 and Bcl-xL proteins.