Reconstructed human epidermis (RHE) is used in non-animal testing for hazard analysis and reconstructed human skin (RHS) gains growing interest in preclinical drug development. RHE and RHS have been characterized regarding their barrier function, but knowledge about biotransformation capacity in these constructs and in human skin remains rather poor. However, metabolizing enzymes can be highly relevant for the efficacy of topical dermatics as well as genotoxicity and sensitization. We have compared the esteratic cleavage of the prednisolone diester prednicarbate and the enzyme kinetic parameters (V(max) and S(0.5)) of the model substrate fluorescein diacetate (FDA) in commercially available RHS and RHE with excised human skin and monolayer cultures of normal and immortalized human keratinocytes and of fibroblasts. Formation of the main metabolite prednisolone and of fluorescein ranked as: RHS ∼ RHE > excised human skin and keratinocytes > fibroblasts, respectively. Because of the aromatic probe, however, V(max) of FDA cleavage did not show a linear relationship with prednicarbate metabolism. In conclusion, RHE and RHS may be useful to quantitatively address esterase activity of human skin in drug development and hazard analysis, although an increased activity compared to native human skin has to be taken into account.