Hydrocortisone (HC) penetration into excised human skin from 1% (w/w) HC containing formulations with white petrolatum, an o/w cream and an aqueous polyacrylate gel is described. The experiments were performed with pure HC and the inclusion compounds with beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin. The smallest HC amounts in the skin were found with the petrolatum preparations. No differences between the incorporation of free HC and the inclusion compounds was found. The highest HC amounts in the skin are found with the o/w cream and hydrogel formulation containing nonincluded HC. Incorporation of both inclusion compounds into these vehicles diminishes the HC concentration in the upper skin layers but not in the dermis. It is assumed that the preferred penetration route for the easily soluble inclusion compounds is a transappendageal diffusion rather than a transdermal one. A comparison with release results by using an ointment liberation model gives a good correlation with the penetration results in the dermis but not in the other skin layers.