The dimeric iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila contains one 4Fe-4S center and one FMN per monomer, and is the prototype of a family widely distributed among strictly anaerobic prokaryotes. Although Isf is able to oxidize ferredoxin, the physiological electron acceptor is unknown; thus, the ability of Isf to reduce O2 and H2O2 was investigated. The product of O2 or H2O2 reduction by Isf was determined to be water. The kinetic parameters of the oxidative half-reactions with O2 and H2O2 as electron acceptors were consistent with a role for Isf in combating oxidative stress. Isf depleted of the 4Fe-4S cluster was unable to oxidize ferredoxin and reduce the FMN cofactor, supporting a role for the cluster in transfer of electrons from ferredoxin to the cofactor. The implications of these properties on the possible function and mechanism of Isf are discussed.