Retinoic acid biosynthesis in rabbit liver was catalyzed by cytosolic NAD(+)-dependent dehydrogenase and oxygen-dependent oxidase, with an activity ratio of 59% and 41% in the presence of 2 mM dithiothreitol under aerobic conditions. The two enzymes could be well separated by fractionation involving ammonium sulfate precipitation. Purification of the enzymes indicated that the oxygen-dependent enzyme was a flavoenzyme, retinal oxidase (EC 22.214.171.124), composed of two 135 kDa subunits; and the NAD(+)-dependent enzyme was a basic pI retinal dehydrogenase composed of four 55-kDa subunits. A high concentration (1-2 mM) of DTT was required to stabilize the activity of retinal dehydrogenase during the purification procedures and storage, but inhibited the activity of retinal oxidase by 13-38%. The physiological roles of the two retinoic acid synthases in liver cytosol were discussed.