Abstract The study was performed to demonstrate superoxide radical (O(2).-) generation, systemic inflammation and liver injury caused by heatstroke and to reveal suppressive effects of moderate hypothermia. Heatstroke was defined as achieving pharyngeal temperature of 40 degrees C with arterial pressure reduction. Heatstroke rats were divided to four groups by the temperature after the onset; 40 degrees C, 37 degrees C, 32 degrees C and sham-treated with 37 degrees C. O(2).- current was measured continuously in the right atrium using an electrochemical O(2).- sensor. The O(2).- current increased in all groups except for the sham-treated group during the induction. After the onset of heatstroke, the O(2).- current was suppressed with temperature-dependency. Plasma and liver high-mobility group box 1, intercellular adhesion molecule-1, plasma aspartate aminotransferase and alanine aminotransferase were also suppressed with the suppression of O(2).- generation. Therefore, excessive O(2).- generation might be a key factor in heatstroke and the suppression with moderate hypothermia would be a therapeutic modality.