Our laboratory has investigated the azoreduction of dimethylaminoazobenzene (DAB) and its analogs by hepatic microsomal cytochrome P450. We have extended these studies to the cytosolic fraction of the mammalian liver using the molybdoflavoenzyme, aldehyde oxidase. Purified rabbit liver aldehyde oxidase readily reduced azo dyes which are mainly water soluble and contain charged groups. Lipophilic azo dyes, although readily reduced by microsomal cytochrome P450, were either poor substrates or not reduced at all. Kinetic measurements revealed no relationship between Vmax and Km for all dyes. More extensive studies were conducted on four azo dyes, o-methyl, red, 2'-pyridyl-DAB, sulfonazo III and Orange II, with characteristic functional groups. With each of these substrates, azoreductase activity was greatest when 2-hydropyrimidine (2-OHP) was the electron donor compared to N1-methylnicotinamide (N-MN), propionaldehyde and butyraldehyde. With 2-OHP as the electron donor, o-methyl red and 2'-pyridyl DAB exhibited maximal activity at pH 5.0 while sulfonazo III and Orange II showed maximal activity at pH 9.5 and 7.0, respectively. Km values for o-methyl red and 2'-pyridyl DAB were lower at their pH optima whereas that for sulfonazo III was higher at its pH optimum. There was also no correlation between maximal activity and Km; apparently Km is not a primary determinant for activity. The degree of ionization of function groups depends on pH. Since highest activity is seen at that pH in which maximal ionization of the substrate occurs, it can be concluded that rate of reduction is at least partially dependent on the charged state of the substrate. Azoreduction was inhibited by menadione and SKF 525-A. Sensitivity to inhibition by menadione was greatest at the pH where 2-OHP exhibited considerably higher activity than N-MN, but no differential was seen at the pH where activities with the two-electron donors were similar. On the other hand, sensitivity of azoreductase activity to inhibition by SKF 525-A was the same irrespective of electron donor, indicating that the mechanisms for these two inhibitors were different.