All-trans-retinol is the precursor for all-trans-retinoic acid, the activating ligand for nuclear transcription factors retinoic acid receptors. In the cytosol of various cells, most retinol exists in a bound form, complexed with cellular retinol binding protein type I (holo-CRBP). Whether retinoic acid is produced from the free or bound form of retinol is not yet clear. Here, we present evidence that holo-CRBP is recognized as substrate by human microsomal short-chain dehydrogenase/reductase (SDR) RoDH-4 with the K(m) value close to the liver concentration of holo-CRBP. The ability to utilize holo-CRBP differentiates RoDH-4 from a related enzyme, RoDH-like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), which is 3-fold more active with free retinol than RoDH-4 but is 15-fold less active toward holo-CRBP. Recognition of the cytosolic holo-CRBP as substrate is consistent with RoDH-4 orientation in the membrane. As established by immunoprecipitation and glycosylation scanning, RoDH-4 faces the cytosolic side of the membrane. Purified RoDH-4, stabilized by reconstitution into proteoliposomes, exhibits the apparent K(m) values for substrates and NAD(+) similar to those of the microsomal enzyme and oxidizes holo-CRBP with the catalytic efficiency (k(cat)/K(m)) of 59 min(-1) mM(-1). Apo-CRBP acts as a strong competitive inhibitor of holo-CRBP oxidation with an apparent K(i) value of 0.2 microM. The results of this study suggest that the human retinol-active SDRs are not functionally equivalent and that, in contrast to RoDH-like 3alpha-HSD, RoDH-4 can access the bound form of retinol for retinoic acid production and is regulated by the apo-/holo-CRBP ratio.