Pyruvate formate-lyase activating enzyme utilizes an iron-sulfur cluster and S-adenosylmethionine to generate the catalytically essential glycyl radical on pyruvate formate-lyase. Variable-temperature (4.2-200 K) and variable-field (0.05-8 T) Mössbauer spectroscopy has been used to characterize the iron-sulfur clusters present in anaerobically isolated pyruvate formate-lyase activating enzyme and in the dithionite-reduced form of the enzyme. Detailed analysis of the Mössbauer data indicates that the anaerobically isolated enzyme contains a mixture of Fe-S clusters with the cuboidal [3Fe-4S] + clusters as the primary cluster form, accounting for 66% of the total iron. Other forms present include [2Fe-2S] 2+ (12% of total Fe) and [4Fe-4S] 2+ (8% of total iron). Careful examination of Mössbauer spectra recorded at various applied fields reveal a fourth spectral component which is assigned to a linear [3Fe-4S] + (∼10% of total Fe). Reduction of the as-isolated enzyme by dithionite, interestingly, converts all cluster types into the [4Fe-4S] form with a mixture of 2+ (66% of total iron) and 1+ (12% of total iron) oxidation states. These results are discussed in light of the proposed role for the iron-sulfur cluster in radical generation.