Integral and peripheral forms of a microsomal retinol dehydrogenase (RoDH) have been distinguished in rat liver through differences in solubility, behavior toward affinity resins, and phase partitioning with Triton X-114. Despite physical differences, polyclonal antibodies raised against integral RoDH recognized peripheral RoDH. No obvious differences were observed in substrate specificity between the two forms. Integral and peripheral RoDH catalyzed retinal synthesis from all-trans-retinol bound to cellular retinol-binding protein, type I (CRBP), with similar Km values of 0.6 and 0.4 microM, respectively. Both also discriminated against CRBP-bound all-trans-3,4-didehydroretinol and against 9-cis-retinol. Phenylarsine oxide inhibited both forms with IC50 values of 5 microM (integral) and 15 microM (peripheral). The more stable peripheral form has been reduced to two major polypeptides that migrate as 34 and 54 kDa bands on SDS-PAGE. The active site of this form has been associated with the 34 kDa polypeptide by covalent binding and inactivation with phenylarsine oxide and by cross-linking to holo-CRBP. Cross-linking required cofactor and was maximum with NADP, consistent with the ordered bisubstrate reaction mechanism of an NADP-supported dehydrogenase. The 34 kDa polypeptide has a subunit molecular weight and other attributes typical of short-chain alcohol dehydrogenases (SCAD) including the highly-conserved SCAD sequence WXLVNNAG, Zn2+ independence; inhibition by carbenoxolone (IC50 = 55 microM), and insensitivity to inhibition by ethanol and 4-methylpyrazole. Tight association between the 34 and 54 kDa polypeptides was demonstrated by their coelution through several columns and the precipitation of RoDH activity with either anti-34 kDa or anti-54 kDa antisera. Because SCAD normally occur as homomultimers, however, the 54 kDa polypeptide is not likely to be a subunit of the peripheral form. This work provides new evidence that the retinol-CRBP "cassette" serves as a substrate for a microsomal RoDH and further characterizes the RoDH.