Electron paramagnetic resonance spectroscopy was used to directly measure free radical generation in perfused rabbit hearts. Hearts were freeze-clamped at 77 degrees K during control perfusion, after 10 min of normothermic global ischemia (no coronary flow), or following post-ischemic reperfusion with oxygenated perfusate. The spectra of these hearts exhibited three different signals with different power saturation and temperature stability: signal A was isotropic with g = 2.004; signal B was anisotropic with axial symmetry with g parallel = 2.033 and g perpendicular = 2.005; signal C was an isotropic triplet with g = 2.000 and hyperfine splitting an = 24 G (1 G = 0.1 mT). The g values, linewidth, power saturation, and temperature stability of signal A are identical to those of a carbon-centered semiquinone, whereas those of signal B are similar to alkyl peroxyl or superoxide oxygen-centered free radicals; signal C is most likely a nitrogen-centered free radical. In the control heart samples signal A predominated, whereas in ischemic hearts signal A decreased in intensity, and signals B and C became more intense; with reperfusion all three signals markedly increased. Free radical concentrations derived from the intensities of the B and C signals peaked 10 sec after initiation of reflow. At this time the oxygen-centered free radical concentration derived from the intensity of signal B was increased over six times the concentration measured in control hearts and over two times the concentration measured in ischemic hearts. Hypoxic reperfusion did not increase any of the free radical signals over the levels observed during ischemia. These experiments directly demonstrate that reactive oxygen-centered free radicals are generated in hearts during ischemia and that a burst of oxygen radical generation occurs within moments of reperfusion.