A method was designed to detect free radicals in circulating blood of animals using extracorporeal circulation and electron paramagnetic resonance (EPR). An arterial-venous shunt was inserted into male Wistar rats between the distal abdominal aorta and the inferior vena cava. The polyethylene tube of the shunt was flowed through a quartz EPR cell. The circulation was supported by manual pumping and maintained at a rate of 3 ml/min. No signal was detected in the blood of untreated rats. After a bolus injection of four spin labels, 2,2,6,6-tetramethyl-piperidine-1-oxyl, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-pyrroline-1-oxyl, signals from each were detected. The intensity of the signal decreased exponentially and the half time was 23.9, 21.6, 47.1, 42.4 sec, respectively. After administration of ascorbic acid, ascorbyl radical signals were detected. This was enhanced by ethylenediaminetetraacetic acid-Fe or diethylenetriaminepentaacetic acid-Fe but not desferrioxamine-Fe. This assay system is useful in detecting free radicals either administered or generated in vivo.