Aim and Methods: Drug development in dermatotherapy and also development of transdermal therapeutic systems (TTS) demand high-predictive in vitro models to estimate drug levels in skin and systemic uptake. Here we compare three ready-to-use models, reconstructed human epidermis, split porcine skin and the perfused porcine forelimb. 17β-Estradiol (E₂), which is highly metabolized by skin cells, serves as model drug since E₂ application is of high relevance in hormone replacement therapy while topical E₂ may promote wound healing. E₂ TTS, gel and an ethanolic solution were investigated for cutaneous penetration, permeation and metabolism. Results: E₂ TTS enabled an E₂ uptake of 42.9% of the applied dose accompanied by a high percentage of E₂ metabolism (30% of the penetrated dose) in the perfused porcine forelimb. In Franz cell experiments with reconstructed human epidermis and split porcine skin, the gel allowed an E₂ uptake of 41.7 and 22.9% of the applied dose accompanied by a high E₂ metabolism (42.6 and 28.6% of the penetrated dose). Due to toxic effects of the vehicle, this was not true with an ethanolic solution, then E₂ permeation and metabolism were clearly diminished. Most importantly, the in vitro models proved to be predictive with respect to the E₂/estrone ratio in female plasma under transdermal hormone replacement therapy. Conclusion: In vitro tests should reduce the need for both animal and human studies for cutaneous uptake and metabolism in the future.