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The aim of the present study was to use the in vitro human skin sandwich system in order to quantify the influence of formulation variables on intrafollicular hydrocortisone permeation. The investigated variables were the pH and the viscosity of the topical formulation as well as the presence of chemical enhancers (carvone, menthone, oleic acid and sodium lauryl sulphate). Furthermore, skin sandwich hydration was also varied in order to determine if the method itself can be run using only partially hydrated skin tissues. It was determined that the follicular contribution to hydrocortisone flux decreased marginally with increasing alkalinity in the pH range 3–8.8. Intrafollicular penetration was markedly reduced when HPMC gels were used instead of an aqueous solution. Pretreating the skin with chemical enhancers also reduced the follicular contribution to flux, probably due to permeabilisation of the continuous stratum corneum. Furthermore, it was not possible to satisfactorily modify the skin sandwich method so that it could be deployed using less hydrated skin.