The permeation of estradiol from vesicular formulations through human stratum corneum was studied in vitro. The vesicles were composed of nonionic n-alkyl polyoxyethylene ether surfactants (CnEOm). The thermodynamic activity of estradiol present in each formulation was kept constant by saturating all formulations with estradiol. The effects of both the particle size and the composition of the formulation on estradiol permeation across excised human stratum corneum were investigated. Stratum corneum that was pretreated with empty surfactant carriers allowed for significantly higher estradiol fluxes compared with untreated stratum corneum. However, estradiol fluxes obtained in these pretreatment experiments appeared to be significantly lower than those obtained by the direct application of the estradiol-saturated carrier formulation on top of the stratum corneum. Furthermore, in the case of pretreatment of the stratum corneum, an increase in carrier size resulted in a decrease in estradiol flux. For direct application the opposite was found. Two mechanisms are proposed to play an important role in vesicle-skin interactions, i.e., the penetration enhancing effect of surfactant molecules and the effect of the vesicular structures that are most likely caused by adsorption of the vesicles at the stratum corneum-suspension interface.