Both oxidative stress and aberrantly high cytoplasmic Ca2+ levels can result in cytotoxicity induced by heat via activation of the apoptotic cell death program ; however, the precise mechanisms by which heat stress induces apoptosis are poorly defined. Furthermore, mitochondria play an essential role in regulating apoptosis and cell death in response to numerous cytotoxic insults, including heat stress, via sensing oxidative stress as well as integrating and transducing the stress signal. It has been reported that cytoplasmic Ca2+ overload can result in cytotoxicity, concomitant with activation of the intrinsic, or mitochondriadependent, apoptotic pathway. However, whether apoptosis of endothelial cells occurs in response to heat stress, subsequent oxidative stress, altered calcium signaling, or a combination thereof, remains to be investigated. The objective of the present study was to explore mechanisms of heat stressinduced apoptosis in HUVEC cells. We hypothesized that heat stress-induced cytotoxicity would occur concomitant with increases in apoptotic markers, including upregulation or activation of pro-apoptotic proteins and nucleosomal DNA fragmentation. In addition to its effects on apoptosis, we also found that heat stress triggered the unfolded protein response in order to protect cells against ER stress, although this early response declined following the cessation of heat stress. Furthermore, we demonstrate that heat stress-induced apoptosis in HUVEC cells proceeds through the calcium-mediated mitochondrial apoptotic pathway, with ROS acting upstream in this process. Finally, we show that the elevation of cytoplasmic Ca2+ following heat stress is mediated in part through upregulation of IP3R. Heat is the most fundamental factor in the pathogenesis of heat stroke and can be directly toxic to cells. Temperature elevation can result in vascular endothelium injury, and it has been reported that the endothelial cell is the primary cell PI-103 PI3K inhibitor population affected during severe heat stroke. The endothelial cell also is an early target in heat stress injury, thus the mechanisms of endothelial cell injury and cell death are highly relevant to understanding the pathogenesis of heat stroke. Our earlier clinical trials found that patients with severe heat stroke present with serious vascular endothelial cell injury, and previous studies in HUVEC cells found that inhibition of endothelial cell proliferation directly contributed to the cytotoxic effects of heat stress. Our previous work also confirmed that endothelial cell apoptosis may be mechanistically relevant to the pathogenesis of heat stroke. Here, we expand our investigation to the temperature- and time-dependent effects of heat stress on endothelial cell apoptosis, including the relevant SB431542 signaling pathways, upstream signaling molecules and cross-talk between signaling intermediates.