Penetration and permeation of topically applied substances may be studied in vitro either by using excised human and animal skin or with a 3-dimensional artificial skin construct (ASC). In the present study, ASC consisting of dermal and epidermal layers were cultivated with human dermal fibroblasts and spontaneously transformed human epidermal cells from the HaCaT-cell line. For comparative purposes the permeation barrier of ASC was also evaluated using a commercial skin model (AST-2000). Higher drug permeabilities were achieved with ASC in comparison with excised human stratum corneum (EHSC). The factors between permeation coefficients of ASC and EHSC depend on the respective drugs and also on different types of the formulation. Due to higher permeation coefficients obtained, the running time of the experiments could be reduced in the case of ASC. A further advantage of ASC is the independency of skin donations. Additionally, cultivation of ASC for the use in permeation studies is possible in advance, because storage conditions under nitrogen freezing do not affect ASC quality negatively.