Hypothermic perfusion of the heart decreases oxidative phosphorylation and increases NADH. Because O(2) and substrates remain available and respiration (electron transport system, ETS) may become impaired, we examined whether reactive oxygen species (ROS) exist in excess during hypothermic perfusion. A fiberoptic probe was placed on the left ventricular free wall of isolated guinea pig hearts to record intracellular ROS, principally superoxide (O(2)(-).), and an extracellular reactive nitrogen reactant, principally peroxynitrite (ONOO(-)), a product of nitric oxide (NO.) + O(2)(-). Hearts were loaded with dihydroethidium (DHE), which is oxidized by O(2)(-). to ethidium, or were perfused with l-tyrosine, which is oxidized by ONOO(-) to dityrosine (diTyr). Shifts in fluorescence were measured online; diTyr fluorescence was also measured in the coronary effluent. To validate our methods and to examine the source and identity of ROS during cold perfusion, we examined the effects of a superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME), and several agents that impair electron flux through the ETS: menadione, sodium azide (NaN(3)), and 2,3-butanedione monoxime (BDM). Drugs were given before or during cold perfusion. ROS measured by DHE was inversely proportional to the temperature between 37 degrees C and 3 degrees C. We found that perfusion at 17 degrees C increased DHE threefold versus perfusion at 37 degrees C; this was reversed by MnTBAP, but not by l-NAME or BDM, and was markedly augmented by menadione and NaN(3). Perfusion at 17 degrees C also increased myocardial and effluent diTyr (ONOO(-)) by twofold. l-NAME, MnTBAP, or BDM perfused at 37 degrees C before cooling or during 17 degrees C perfusion abrogated, whereas menadione and NaN(3) again enhanced the cold-induced increase in ROS. Our results suggest that hypothermia moderately enhances O(2)(-). generation by mitochondria, whereas O(2)(-). dismutation is markedly slowed. Also, the increase in O(2)(-). during hypothermia reacts with available NO. to produce ONOO(-), and drug-induced O(2)(-). dismutation eliminates the hypothermia-induced increase in O(2)(-).