Previous work established that increased expression of heat shock proteins (HSPs) in the vessel wall might evoke proinflammatory and autoimmune reactions in the pathogenesis of atherosclerosis. The present study was designed to further scrutinize the molecular mechanisms of HSP expression involving activation of heat shock transcription factors (HSFs) in atherosclerotic lesions in animal models. Severe atherosclerotic lesions developed in the aortas of rabbits 16 weeks after feeding a 0.2% cholesterol diet. When protein extracts from the aortas were subjected to Western blot analysis, the level of HSF1 in proteins from atherosclerotic lesions of hypercholesterolemic rabbits were significantly higher than those of normal vessels. Gel mobility shift assays revealed the formation of protein-heat shock element complexes containing HSF1 in protein extracts from atherosclerotic lesion. Furthermore, triglyceride-rich lipoprotein, oxidized-triglyceride-rich lipoprotein, low-density lipoprotein, and oxidized low-density lipoprotein did not activate HSF1 in cultured smooth muscle cells, whereas HSF1 was highly activated in cells treated with tumor necrosis factor-alpha. Interestingly, mechanical stretching of smooth muscle cells resulted in HSF1 translocation from the cytoplasm to the nucleus and hyperphosphorylation followed by increased HSP70 expression. Thus, our findings provide the first evidence that HSF1 is activated and highly expressed in atherosclerotic lesions and that cytokine stimulation and disturbed mechanical stress to the vessel wall may be responsible for such activation.