Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43°C for 20 min) resulted in inhibition of H 2 O 2 -induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H 2 O 2 -induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homooligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H 2 O 2 -induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.Apoptosis signal-regulating kinase 1 (ASK1) is a widely expressed serine-threonine kinase that was initially discovered as a mitogen-activated protein kinase kinase kinase (MAPKKK) in the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK signaling cascades (12, 13). ASK1 functions in apoptosis induced by diverse stimuli, including tumor necrosis factor alpha (TNF-␣), Fas, and many apoptotic stresses (12,13,30). Overexpression of a dominantnegative mutant of ASK1 thus prevents apoptosis induced by withdrawal of growth factors, DNA-damaging agents, TNF-␣, or agonistic antibodies to Fas (4,13,16,30). Activation of ASK1 is reported to induce apoptotic cell death by triggering mitochondrial events that include the release of cytochrome c from mitochondria and the subsequent activation of caspase 9 and caspase 3 (11).Many cellular stresses that stimulate the stress-activated MAPK pathways can also induce expression of heat shock proteins. Heat shock protein 72 (Hsp72) is the major inducible heat shock protein (35). It plays a role in many cellular activities including protein synthesis, folding, and translocation into organelles as well as the assembly of multiprotein complexes (2,5,24,25,33). Hsp72 contains two conserved domains, an ATP-binding domain (ABD) and a peptide-binding domain (PBD), that are important for its chaperon function (7, 21). Hsp72 also prevents cell death initiated by various apoptotic stresses including heat shock, ceramide, ionizing irradiation, TNF-␣, and ischemia (8,14,20,23,32). Hsp72 suppresses several apoptotic signaling pathways, including caspase cascades and stress-activated MAPK pathways that include the JNK and p38 signaling cascades (3,8,9,15,17,19,22,23,29,31). Furthermore, Hsp72 has been shown to physically interact with and inhibit Apaf-1 and apoptosis-inducing factor, resulting in suppression of caspase-dependent and -independent apoptosis, respectively ...