In the present study the influence of biotin on human cultured keratinocytes was examined. The prior-ranking aim was to characterize the effect mechanism and the cellular reaction of the keratinocytes to different drug concentrations. The human epidermal cells have shown a high tolerance of the vitamin. Both the proliferation and the differentiation of the keratinocytes can be judged to be free of the influence of biotin. No regulatory influence on the apoptosis, which was induced by fatty acids, could be detected. After exceeding a necessary swelling concentration an increasing effect of biotin on the lipid synthesis in the kerationcytes was provably. Following the results from the cell culture and the known necessary effective concentration of biotin we determined the penetration behavior of topical applied biotin into human skin ex vivo. Within these examinations the main interest lay on the systematic modulation of the penetration behavior. So we compared the biotin penetration from standardized emulsions with modulated emulsions and with different types of biotin containing colloidal drug carriers too. Our results particularly recommend the use of the modulators hyaluronidase and urea for an effective enhancement of biotin into the living layers of the skin.