Heat shock protein 70 (Hsp70) is a well-known inhibitor of apoptotic pathways; however, a role for Hsp70 in the modulation of death receptor-mediated apoptosis remains largely unexplored. In this study, the ability of Hsp70 to modulate tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis was examined in SW480 and CCRF-CEM cells. These lines exhibit the characteristics of type I cells (SW480, human colon adenocarcinoma), with no requirement for mitochondrial involvement to exhibit apoptosis following death receptor engagement and type II cells (CCRF-CEM, human leukemic T cell), which do require amplification of the signal through the mitochondria. Unexpectedly, expression of Hsp70 in the type II CCRF-CEM cells enhanced the extent of TRAIL-induced apoptosis, but in SW480, Hsp70 had no impact on TRAIL-induced apoptosis. The enhanced TRAIL-induced apoptosis was accompanied by an up-regulation of TRAIL receptors, R1 and R2, at the cell surface as determined by flow cytometry and at the transcriptional level as assessed by real-time polymerase chain reaction (PCR). Increased expression of Hsp70 led to up-regulated expression of p53, and chromatin immunoprecipitation combined with real-time PCR revealed increased binding of p53 to its consensus sequence in the TRAIL-R2 gene. In contrast, expression of Hsp70 in SW480 cells did not increase p53 or TRAIL-R1 or TRAIL-R2 surface expression. This result is in marked contrast to most apoptotic stresses, including TNFalpha and Fas ligand, where Hsp70 has been shown to inhibit apoptosis in type II cells. These findings suggest that in tumors retaining functional p53 and expressing high levels of Hsp70, TRAIL may be an effective therapy.