ABSTRACT We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum , a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The Δ pyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF , creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta , which encodes the enzyme phosphotransacetylase. The C. thermocellum Δ pta strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in C. thermocellum , a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources.