Hypothermia decreases ischemia‐reperfusion (IR) injury. IR injury has been associated with mitochondrial permeability transition pore (PTP) opening, and decreased activity of the electron transport chain. We studied mitochondria from isolated hearts subjected to normothermic and hypothermic ischemia. Guinea pig isolated hearts were perfused at constant pressure with Krebs‐Ringer's solution at 37 ºC and subjected to 30 min of ischemia at 37ºC or 17ºC. After 5 min of reperfusion mitochondria were isolated. Mitochondrial [Ca2+]m, pH, membrane potential (ΔΨm) and NADH were measured spectrophotometrically with indo 1, BCECF, rhodamine 123 and autofluorescence. After energizing with pyruvic acid, [CaCl2] (0‐100 μM) and ADP were added. Adding CaCl2 caused a dose dependent [Ca2+]m increase. Ca2+ induced PTP opening, assessed by collapse of ΔΨm, and prevented by cyclosporine A, occurred after adding 50 μM CaCl2 after 37ºC IR, and adding 100 μM CaCl2 after 17ºC IR. ADP decreased ΔΨm, pH and NADH and increased [Ca2+]m in all mitochondria. These bioenergetic markers recovered after 17ºC IR, but not after 37ºC IR. Our study shows that hypothermia protects mitochondria against IR injury as shown by resistance to Ca2+ induced PTP opening and appropriate responses to ADP. This study emphasizes the importance of mitochondrial function in protecting against IR injury, and gives insight into how hypothermia reduces IR injury.