Previous studies have demonstrated that Geobacter sulfurreducens requires the c-type cytochrome OmcZ, which is present in large (OmcZ L ; 50-kDa) and small (OmcZ s ; 30-kDa) forms, for optimal current production in microbial fuel cells. This protein was further characterized to aid in understanding its role in current production. Subcellular-localization studies suggested that OmcZ s was the predominant extracellular form of OmcZ. N- and C-terminal amino acid sequence analysis of purified OmcZs and molecular weight measurements indicated that OmcZ s is a cleaved product of OmcZL retaining all 8 hemes, including 1 heme with the unusual c-type heme-binding motif CX 14 CH. The purified OmcZ s was remarkably thermally stable (thermaldenaturing temperature, 94.2°C). Redox titration analysis revealed that the midpoint reduction potential of OmcZ s is approximately - 220 mV (versus the standard hydrogen electrode [SHE]) with nonequivalent heme groups that cover a large reduction potential range (- 420 to - 60 mV). OmcZ s transferred electrons in vitro to a diversity of potential extracellular electron acceptors, such as Fe(III) citrate, U(VI), Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analogue anthraquinone-2,6-disulfonate, but not Fe(III) oxide. The biochemical properties and extracellular localization of OmcZ suggest that it is well suited for promoting electron transfer in current-producing biofilms of G. sulfurreducens.