A nonreplicative plasmid containing an internal aad gene fragment has been integrated into the chromosome of Clostridium acetobutylicum ATCC 824. Transformation was accomplished by electroporation with relatively high concentrations of methylated plasmid DNA. Southern hybridization experiments revealed that integration occurred by single crossover homologous recombination inactivating the aad gene. Integrants were relatively stable after 25 generations. Inactivation of the aad gene drastically reduced solvent production. This result suggests that aldehyde/alcohol dehydrogenase (AAD) plays a important role in butanol production.