Trimethylamine dehydrogenase is a bacterial enzyme which contains two redox centers: a flavin mononucleotide (FMN) group which constitutes the active site and a [4Fe-4S](1+,2+) cluster which transfers the electrons provided by the FMN to an electron-transferring flavoprotein. According to the x-ray crystal structure, the renter-to-center distance is equal to 12 Angstrom and the nearest atoms of the two centers are separated by a 4 Angstrom gap. Although this arrangement does not appear especially favorable for mediating strong magnetic interactions, a triplet state electron paramagnetic resonance (EPR) spectrum arising from the intercenter magnetic coupling is observed at X band (9 GHz) when the enzyme is reduced by its substrate. In earlier work, the temperature dependence of this spectrum and its analysis based on a tripler state spin Hamiltonian were used to propose the range (0.8-100 cm(-1)) for the parameter J(0) of the isotropic interaction J(0)S(A).S-B, but neither the magnitude of J(0) nor its sign could be further specified [R. C, Stevenson, W. R, Dunham, R. H. Sands, T. P. Singer, and H. Beinert, Biochim. Biophys. Acta 869, 81 (1986)]. In the present work, we have studied the interaction EPR spectrum in the range 9-340 GHz, Numerical simulations based on a spin Hamiltonian describing a system of two S=1/2 interacting spins allowed us to determine the full set of parameters describing the magnetic interactions between the FMN radical and the [4Fe-4S](1+) cluster. In particular, our study demonstrates that the coupling is antiferromagnetic with J(0) = + 0.72 cm(-1). Although this value corresponds to the lower limit of the range proposed previously, it still appears markedly larger than those measured in biological systems in which a similar arrangement of two paramagnetic centers is found.