The biochemical mechanisms for growth tolerance to a 100% CO headspace in cultures, and butanol plus ethanol production from CO by Butyribacterium methylotrophicum were assessed in the wild-type and CO-adapted strains. The CO-adapted strain grew on glucose or CO under a 100% CO headspace, whereas, the growth of the wild-type strain was severely inhibited by 100% CO. The CO-adapted strain, unlike the wildtype, also produced butyrate, from either pyruvate or CO. The CO-adapted strain was a metabolic mutant having higher levels of ferredoxin-NAD oxidoreductase activity, which was not inhibited by NADH. Consequently, only the CO-adapted strain can grow on CO because CO oxidation generates reduced ferredoxin which, via the mutated ferredoxin-NAD reductase activity, forms reduced NADH required for catabolism. When the CO-adapted strain was grown at pH 6.0 it produced butanol (0.33 g/l) and ethanol (0.5 g/l) from CO and the cells contained the following NAD-linked enzyme activities (mu mol min(-1) mg protein(-1)): butyraldehyde dehydrogenase (227), butanol dehydrogenase (686), acetaldehyde dehydrogenase (82) and ethanol dehydrogenase (129).