To replace animal experiments for the risk assessment in cutaneous absorption European and Non-European regulatory Authorities urge to develop in vitro test systems. A variety of methods have been established which, however, vary in several respects, such as the nature of the barrier and used media. Since both affect drug uptake we have compared the influence of albumin (BSA) in the acceptor medium on the permeation and metabolism of highly lipophilic steroidal drugs, testosterone and prednicarbate (PC). Surprisingly, the addition of BSA to the acceptor medium slightly reduced the steroid permeation, especially when formulations of poor PC uptake were tested. Moreover, with slow drug permeation the metabolite pattern changed as compared to PC metabolites to be found in albumin-free acceptor medium. This was clearly less the case with PC incorporated into newly developed solid lipid nanoparticles accelerating PC uptake about fourfold. The penetration of testosterone was not influenced by BSA in the acceptor medium. Summarising, these results contribute essentially to the development of appropriate in vitro methods for testing of the cutaneous absorption of drugs, ingredients of cosmetics and for the risk assessment of xenobiotics, pesticides and biozides.