AbstractMechanistic aspects of the mode of action of the soluble monooxygenase system ofMethylosinus trichosporiumOB3b have been investigated. The hydroxylation of 4‐deuteroanisole in the 4‐position was shown to proceed via an NIH shift with a deuterium retention of 66 %, indicative of an epoxide intermediate. Isotopic studies using ethylbenzene and ethylbertzene‐d10showed an isotopic effect,kH/kD= 4.5 for benzylic hydroxylation, and an inverse isotopic effect,kH/kD= 0.75 for arene hydroxylation. It is suggested that a stepwise mechanism (hydrogen abstraction and hydroxylation) could be in