A dual-probe microdialysis technique was developed for the simultaneous monitoring of glucose and related metabolite levels during testicular ischemia in rats. The determinations of lactate and pyruvate were achieved by liquid chromatography within 5 min. Glucose was determined by a microdialysis analyzer. A unilateral ligation was produced by occlusion of the right artery for 2 or 4 h in anesthetized rats. Microdialysis probes were inserted in both sides of the testis to simultaneously monitor glucose, lactate and pyruvate during basal, ischemia and reperfusion periods. Dynamic and comparative changes in these analytes in ipsilateral and contralateral testes were demonstrated. The present technique can be used as a tool for exploring energy related metabolites and their relationships in testicular ischemia.