Cosmetics containing rhododendrol (RD) were voluntarily recalled after incidents of leukoderma related to their use. Users reported using up to five different RD-containing products by layered application. In this study, we investigated the effects of layered application, formulations, and their components on the skin permeation of cosmetics containing RD. Experiments were designed to simulate actual in-use conditions, such as varying application volumes, physical mixing of formulations, sequence of cosmetics application and time interval between applications, to establish their effect on the skin permeation of RD. Milk and lotion RD-containing cosmetics (2%), 1% aqueous RD, and preparations of formulation components were applied as the first or second layers as finite doses of 10 or 20 µL/cm 2. Permeation experiments were performed through excised porcine ear skin using Franz diffusion cells with an effective diffusion area of 1.77 cm 2. Cosmetics applied by layered application exhibited lower skin permeation of RD compared with a single application despite having the same application dose. High initial volume (20 µL at 0 or 5 sec) did not exhibit any significant reduction in the permeation of RD. Formulations and their components caused varying reductions in RD permeation, probably due to changes in thermodynamic activity of the active component. Layered application, formulation components, application volume, time interval and sequence of application had significant influences on the skin permeation of the active component. Moreover, this study established a method of investigating the influence of formulations and their components on the skin permeation of actives after layered application.