The presence of a sufficient quantity of a drug in the epidermis is a necessary prerequisite for influencing epidermal proliferation and differentiation processes. With the aim of obtaining a high concentration of a potential active agent (biotin) at the target area after topical application, the influence of the vehicle on biotin release and penetration was investigated. Liberation studies (multi-layer membrane model) showed that over 50% of the biotin in an oil-in-water (O/W) emulsion (64.8 ± 1.9%) and a micro-emulsion (ME, 54.5 ± 2.4%) is released within 300 min, whereas the degree of release from a water-in-oil emulsion does not exceed 16%. For this latter vehicle, the influence of a controlled drug release on the penetration processes in human skin (Franz cell) results in the penetration of only small quantities of the drug into the skin layers (into the horny layer 7.9 ± 2.6%, living epidermis 0.11 ± 0.06%, dermis 0.38 ± 0.31% within 300 min). The application of an O/W emulsion or an ME appears to be more favourable. Both vehicles lead to not only a large reservoir in the horny layer (O/W 26.5 ± 3.5%, ME 26.0 ± 0.8% within 300 min) of the skin but also important epidermal and dermal concentrations (living epidermis: O/W 2.0 ± 0.9%, ME 0.3 ± 0.2%; dermis: O/W 3.4 ± 1.3%, ME 1.6 ± 0.8% within 300 min). The time-dependent concentration profiles in the skin layers suggest that the use of an ME produces an immediate effect, whereas the application of an O/W emulsion results in a delayed and/or long-t