Background: CYP26A1 is a critical enzyme in the metabolism of all-trans-retinoic acid (atRA). Results: CYP26A1 metabolizes atRA stereoselectively to (4S)-OH-RA, which corresponds to atRA orientation within the CYP26A1 active site. Conclusion: Preference for (4S)-OH-RA is conserved in atRA metabolism and in elimination of 4-OH-RA.