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There is growing evidence that the presence of the cell stress protein heat shock protein (HSP) 60 in the circulation is associated with risk of coronary heart disease. In this study, we measured the association between plasma HSP60 and carotid arterial stiffness in middle-aged men and women.Six hundred and forty-seven men and women aged 50–72 years and free of cardiovascular disease and medication were tested. Carotid artery distensibility coefficient was assessed ultrasonically as a measure of arterial stiffness, and plasma HSP60 was assessed using a sensitive immunoassay.We found a significant, independent association between high plasma levels of HSP60 and increased carotid stiffness. Carotid distensibility coefficient was also related to diabetes, adiposity, blood pressure, lipids, plasma interleukin-6 and C-reactive protein. After adjusting for these factors, the odds of HSP60 concentration of at least 1000 ng/ml were 1.79 (95% confidence intervals 1.06–3.04) for participants in the lowest compared with the highest tertile of the distensibility coefficient.HSP60 is a potent activator of vascular endothelial cells and smooth muscle cells. Thus, it is possible that long-term stimulation of these cell populations by blood-borne HSP60 acts to drive blood vessel changes resulting in decreased arterial elasticity.