Spectroscopic studies were carried with a cytochrome P-450 in Moraxella sp., strain GU2, that could grow on guaiacol or 2-ethoxyphenol as the sole source of carbon and energy. The dissociation constant of the guaiacol-cytochrome complex was estimated to 0.15 microM, as determined in vivo or using the cell soluble extract. Cytochrome P-450 could also bind 2-ethoxyphenol, 2-propoxyphenol, and 2-butoxyphenol, and the dissociation constants have been determined in each case. Metyrapone depressed the degradation of guaiacol by whole bacteria, and was bound competitively to guaiacol with a constant of about 0.8 mM. Some catechol was excreted by the bacteria when growing on either guaiacol or 2-ethoxyphenol. Catechol and the other product of guaiacol demethylation, formaldehyde, were further oxidized by the bacteria. All the data available so far are consistent with cytochrome P-450 in Moraxella GU2 as a hydroxylase for the guaiacol side chain, behaving as a nonspecific O-dealkylase with broad specificity for guaiacol and homologous compounds with a longer carbon part in the side chain.