Two binuclear iron-sulfur clusters (designated S-1 and S-2) are present in succinate dehydrogenase in approximately equal concentration to that of flavin. The large difference in their midpoint potentials (0 and -400 mV, respectively, in the soluble enzyme) permits the acquisition of individual electron paramagnetic resonance spectra characterized by nearly identical rhombic g tensors (gz = 2.025, gy = 1.93, gx = 1.905). Spin-coupling between the two centers is manifested by broadening and splitting of spectra of reconstitutively active and inactive succinate dehydrogenase, respectively, as the temperature is lowered; relief of power saturation of Center S-1 spectra on reduction of Center S 2; and observation of half-field ("delta ms = 2") signals in the dithionite-reduced enzyme. Saturation behavior of fully reduced dehydrogenase is consistent with the presence of S-1 and S-2 at equivalent concentrations/molecule. Simulation of the spin-coupled spectra, assuming dipolar interaction, provides information on molecular structure. Electron paramagnetic resonance spectra of the enzyme in 80% dimethylsulfoxide are nearly identical to the characteristic binuclear spectra obtained with adrenodoxin. These data provide additional evidence for binuclear structure of both Center S-1 and S-2. The extremely fast relaxation of Center S-2 at low temperatures would imply either an anomalously small value of J or an alternative relaxation mechanism, possibly due to the coupling between S-1 and S-2.