The 16-line EPR signal from the Mn(III)/Mn(IV) state of Lactobacillus plantarum Mn catalase was studied at three microwave frequencies : S-band (3.0 GHz), X-band (9.2 GHz), and P-band (15.5 GHz). The spectra were simulated using a program that calculated the hyperfine splitting as a perturbation of the Zeeman term to third order. The transition energies were calculated numerically from the perturbation terms rather than from an explicit expression derived from perturbation theory, as has been done previously for spectra from multinuclear Mn complexes. Nine Mn catalase spectra were fit using a nonlinear least-squares minimization routine assuming an axial S = 1/2 system. Axial g and hyperfine matrices were found to fit the spectra well. Second-order perturbation theory was sufficient to fit the X- and P-band spectra, but third-order perturbation terms were necessary to adequately fit the S-band spectrum and give parameters that agreed with those found at the higher frequencies. The EPR parameters found for this biological Mn dimer (g(z) = 1.990, g(x) = g(y) = 2.008, A1z = 104.1 x 10(-4) cm-1, A1x = A1y = 141.6 x 10(-4) cm-1, A2z = 83.5 x 10(-4) cm-1, A2x = A2y = 76.0 x 10(-4) cm-1) compare well with those for synthetic Mn-(III)/Mn(IV) complexes and with estimates by vector projection using literature values of the independent Mn ions. The success of the simulation method employed here, which can accommodate rhombic systems and carry the perturbation calculation to third or higher orders, will have utility for simulation of the S2 multiline signal at g = 2 from the O2-evolving complex of higher plants and algae.