We applied in vivo microdialysis techniques in examining the relation between norepinephrine and free radical generation on myocardial ischemic injury. We designed the microdialysis probe holding system, which includes loose fixation of the tube and synchronization of the movement of the heart and the probe. The heart was subjected to myocardiac ischemia for 15 min by occlusion of rat left anterior descending coronary artery. We confirmed typical changes in the electrocardiogram. The hydroxyl free radical (.OH) reacts with salicylate and generates 2, 3- and 2,5-dihydroxybenzoic acid (DHBA), which can be measured electrochemically in picomole quantity by a high-performance liquid chromatographic-electrochemical procedure. After probe implantation, the norepinephrine concentration of dialysate decreased over the first 120 min and then reached an almost steady-state level of 0.15 +/- 0.02 nmol/ml. However, when the heart was reperfused, the levels of norepinephrine and 2,3- and 2,5-DHBA were elevated. In vivo microdialysis techniques permit monitoring of norepinephrine levels and free radical generation in myocardial ischemic injury.