The control of normal epithelial cell growth and differentiation by retinoids (vitamin A and its analogs) may involve, at least in part, cellular retinoid-binding proteins. In the present study the levels of cellular retinoic acid (CRABP)- and retinol (CRBP)-binding proteins were measured in cultured normal human epidermal keratinocytes. To this end cells were cultured under either low calcium (nondifferentiating) or normal calcium (differentiating) conditions; in the latter culture the cells were also separated into nondifferentiated, attached cells, and differentiating, shed, cell populations. Two different techniques [gel filtration and polyacrylamide gel electrophoresis (PAGE)] were used, for both the qualitative and the quantitative determinations of retinoid-binding proteins. Gel filtration analysis on Sephadex G75 columns showed the presence of high-molecular-weight binding sites for retinol and retinoic acid in both nondifferentiated and the differentiating keratinocytes; the nature of these binding proteins is unclear. Free CRABP was found in the cytosol of differentiating cells, but was undetectable in nondifferentiated cells. Using a more sensitive PAGE technique, very low levels of CRABP and CRBP could be detected in nondifferentiated keratinocytes. By this technique it was possible to demonstrate very high levels of CRABP only in differentiating keratinocytes; the CRBP levels were found to be very low in differentiating cells and comparable to the amount found in nondifferentiated cells. On the basis of molecular weight determinations by gel exclusion and by electrophoretic mobility, the CRABP and CRBP of cultured keratinocytes were found to be identical to CRBP and CRABP from human epidermis.