Decreased motility has been correlated with lower solvent yields in fermentations with Clostridium acetobutylicum. A spontaneous mutant of C. acetobutylicum was found to be nonmotile as evidenced by bright-field microscopy and motility-agar plates. The loss of motility was accompanied by the production of an altered flagellin. The mutant flagellin was much smaller than the wild-type (32 vs 43 kDa), although the NH2-terminal amino acid sequences of both flagellins were identical. This mutant was simultaneously incapable of producing the solvents acetone and butanol. In vitro enzyme activity analyses demonstrated the absence of three enzymes directly involved in solvent production: acetoacetate decarboxylase (EC 4.1.1.4), acetoacetyl-coenzyme A:acetate/butyrate coenzyme A-transferase (EC 2.8.3.9), and NADP-dependent butyraldehyde dehydrogenase (EC 1.2.1.10).