The azoreduction of N,N-dimethyl-4-aminoazobenzene (DAB) and N-methyl-4-amino-azobenzene (MAB) by rat liver microsomes was investigated. It was shown that measurement of azoreduction of DAB and structurally related azo dyes by the conventional method of substrate disappearance required an anaerobic environment since N-demethylated and ring-hydroxylated metabolites formed aerobically interfered with the assay system, producing quantitatively inaccurate results. Oxygen partially, but not totally, inhibited azoreduction of DAB. Glutathione (GSH) inhibited the azoreduction of DAB but stimulated the azoreduction of MAB. Dithiothreitol also stimulated azoreduction of MAB but had little effect on azoreduction of DAB. Para-hydroxymercuribenzoate (PHMB) and N-ethylmaleimide (NEM) blocked titratable microsomal thiol groups and inhibited azoreduction of MAB. However, the inhibitory action of NEM was weak with DAB azoreduction although PHMB was a potent inhibitor. These findings suggest that microsomal azoreduction of DAB and MAB may proceed via different mechanisms, possibly through different species of cytochrome P-450 which have selective dependence upon the sulfhydryl environment.