Expression of IGF-binding protein-3 (IGFBP-3) and IGFBP-5 in human breast cancer cells induces apoptosis and is associated with modulations in Bcl-2 proteins, suggesting that these IGFBPs induce an intrinsic apoptotic pathway. In this study we demonstrate that although both IGFBPs induced the activation of caspase-8 and caspase-9, the expression of IGFBP-5, but not IGFBP-3, sensitized MDA-MB-231 breast cancer cells to the inhibitory effects of TNFalpha. This sensitivity to TNFalpha was associated with a block in nuclear factor-kappaB-mediated cell survival signals. IGFBP-5 expression was also associated with a caspase-8-independent activation of Bid, increased levels of cytosolic second mitochondria-derived activator of caspase (Smac)/direct inhibitor of apoptosis proteins (IAP) binding protein with low pI (DIABLO), and an enhanced phosphorylation of c-Jun N-terminal kinase, both basally and in response to TNFalpha. These results suggest that IGFBP-5 expression may influence extrinsic apoptotic pathways via a differential modulation of downstream cell survival and cell death pathways. Furthermore, although IGFBP-3 and IGFBP-5 share much structural and functional homology, they can modulate distinct apoptotic pathways in human breast cancer cells.