We previously showed that postmortem serum levels of adrenocorticotropic hormone (ACTH) were significantly higher in cases of hypothermia (cold exposure) than other causes of death. This study examined how the human hypothalamic-pituitary-adrenal axis, and specifically cortisol, responds to hypothermia. Human samples: Autopsies on 205 subjects (147 men and 58 women; age 15-98 years, median 60 years) were performed within 3 days of death. Cause of death was classified as either hypothermia (cold exposure, n = 14) or non-cold exposure (controls; n = 191). Cortisol levels were determined in blood samples obtained from the left and right cardiac chambers and common iliac veins using a chemiluminescent enzyme immunoassay. Adrenal gland tissues samples were stained for cortisol using a rabbit anti-human polyclonal antibody. Cell culture: AtT20, a mouse ACTH secretory cell line, and Y-1, a corticosterone secretory cell line derived from a mouse adrenal tumor, were analyzed in mono-and co-culture, and times courses of ACTH (in AtT20) and corticosterone (in Y-1) secretion were assessed after low temperature exposure mimicking hypothermia and compared with data for samples collected postmortem for other cases of death. However, no correlation between ACTH concentration and cortisol levels was observed in hypothermia cases. Immunohistologic analyses of samples from hypothermia cases showed that cortisol staining was localized primarily to the nucleus rather than the cytoplasm of cells in the zona fasciculata of the adrenal gland. During both mono-culture and co-culture, AtT20 cells secreted high levels of ACTH after 10-15 minutes of cold exposure, whereas corticosterone secretion by Y-1 cells increased slowly during the first 15-20 minutes of cold exposure. Similar to autopsy results, no correlation was detected between ACTH levels and corticosterone secretion, either in mono-culture or co-culture experiments. These results suggested that ACTH-independent cortisol secretion may function as a stress response during cold exposure.