The aromatic CO-dependent O-demethylating activity of Clostridium thermoaceticum was evaluated. Secondary aromatic substituent groups (-OH, -CO2H, -CH2OH, and -OCH3) were critical to O demethylation. O-demethylating activities and specificities were similar from cells grown at the expense of different methoxylated aromatic compounds; all O-methyl-grown cells catalyzed the same sequential O demethylation of multi-methoxylated compounds, suggesting that a broad specificity O demethylase was involved in O demethylation. In cell-fractionation studies, CO-dependent O demethylation was catalyzed by membrane-associated components.