The structure and function of the prototypical dinuclear hydrolase, namely, the aminopeptidase from Aeromonas proteolytica (AAP), was probed by EPR spectroscopy of the mono- and dicobalt(II)-substituted derivatives. A new systematic protocol for the interpretation of Co(II) EPR spectra is described and the S = 3/2 Spin states of the Co(II)-substituted forms of the enzyme have been characterized. This protocol allows the simulation of line shape using theoretically allowed g(eff) values corresponding to an isotropic g(real) value. In addition, the gross distortion of EPR Spectra of high-spin S = 3/2 Co(II) ions has been investigated, and the effects of saturation on the line shapes and on simulation-derived spectral parameters are discussed. For [Co-(AAP)], a distinctive EPR signal was observed in which the hyperfine pattern due to Co-59 was not centered on the low-field absorption feature, and the signal could not be simulated as a single species.