Human alcohol dehydrogenases (ADH1 and ADH4) actively use retinoids oxidized at the cyclohexenyl ring (4-oxo-, 4-hydroxy-, and 3,4-didehydro-retinoids), which are functional compounds in several cells and tissues (i.e., in human skin). Remarkably, activities with 4-oxo-retinal and 4-hydroxy-retinol (kcat = 2050 min(-1) for ADH4) are the highest among retinoids, similar to those of the best aliphatic alcohols. Thus, ADH1 and ADH4 provide a metabolic pathway for the synthesis of the corresponding retinoic acids. Tween 80, a widely used detergent in the retinoid activity assay, behaves as a competitive inhibitor. The Km values for all-trans-retinol (2-3 microM), estimated in the absence of detergent, are 10-fold lower than those obtained at the usual 0.02% Tween 80. This suggests a contribution of ADH to retinoid metabolism more relevant than previously expected. However, Tween 80 stabilizes retinoids in water solution and provides a reliable and reproducible assay, suitable for comparing different ADHs and different retinoid substrates.