The recently discovered [2Fe-2S] cluster in mouse liver ferrochelatase has been characterized using UV-vis, EPR, and Mossbauer spectroscopic techniques. Studies are reported here for the recombinant protein purified from an overproducing transformed Escherichia coli strain. A positive correlation is observed between the presence of the [2Fe-2S] cluster and the enzymatic specific activity and demonstrates the necessity of this cofactor. Chemical analysis revealed that the preparations contained up to 1.3 Fe/molecule and indicated a 1:1 stoichiometry between Fe and acid-labile sulfide. The [2Fe-2S] cluster in the as-isolated ferrochelatase exhibits a UV-vis spectrum indicative of a [2Fe-2S]2+ cluster and is EPR-silent. The 8 T Mossbauer spectrum of the 57Fe-enriched as-isolated protein is well simulated by parameters ΔE(Q) = 0.69 ± 0.03 mm/s and δ = 0.28 ± 0.02 mm/s and confirms the presence of a diamagnetic ground state. Upon reduction with sodium dithionite, ferrochelatase shows a near-axial EPR spectrum with g-values of 2.00, 1.93, and 1.91, consistent with a S = 1/4 mixed valent Fe3+-Fe2+ cluster. The Orbach temperature dependence of the EPR line widths was used to provide an estimate of the exchange coupling J, which was determined to be in the order of 500-650 cm-1 (+JS1.S2 model). Redox titrations monitored by UV-vis and EPR spectroscopy revealed midpoint potentials of -390 ± 10 and -405 ± 10 mV, respectively. Mossbauer spectra of the sodium dithionite-reduced 57Fe-enriched ferrochelatase collected at 4.2 K in the presence of magnetic fields of 60 mT and 8 T strengths were analyzed in the mixed-valent S = 1/4 ground state. Parameters for the ferric site are ΔE(Q) = 1.2 ± 0.2 mm/s and δ = 0.28 ± 0.03 mm/s, with somewhat anisotropic hyperfine splittings; for the ferrous site, ΔE(Q) = 3.3 ± 0.1 mm/s and δ = 0.67 ± 0.04 mm/s with anisotropic hyperfine splittings characteristic of high-spin ferrous ion. The similarities and differences with other characterized [2Fe-2S]+ cluster-containing proteins are discussed.