This study shows that Geobacter sulfurreducens grows on carbon monoxide (CO) as electron donor with fumarate as electron acceptor. Geobacter sulfurreducens was tolerant to high CO levels, with up to 150 kPa in the headspace tested. During growth, hydrogen was detected in very slight amounts (similar to 5 Pa). In assays with cell-free extract of cells grown with CO and fumarate, production of hydrogen from CO was not observed, and hydrogenase activity with benzyl viologen as electron acceptor was very low. Taken together, this suggested that CO is not utilized via hydrogen as intermediate. In the presence of CO, reduction of NADP(+) was observed at a rate comparable to CO oxidation coupled to fumarate reduction in vivo. The G. sulfurreducens genome contains a single putative carbon monoxide dehydrogenase-encoding gene. The gene is part of a predicted operon also comprising a putative Fe-S cluster-binding subunit (CooF) and a FAD-NAD(P) oxidoreductase and is preceded by a putative CO-sensing transcription factor. This cluster may be involved in a novel pathway for CO oxidation, but further studies are necessary to ascertain this. Similar gene clusters are present in several other species belonging to the Deltaproteobacteria and Firmicutes, for which CO utilization is currently not known.