Microorganisms play an important role in the pathomechanism of acne vulgaris which is treated with antibiotics, particulary erythromycin (ERY). The main problem in the topical use of ERY lies in achieving sufficient penetration of the drug into sebaceous follicles. Doubly enhanced penetration of an ion pair composed of ERY and octadecansulfonate (OS) in contrast to the commonly used ERY base was observed, using a multilayer membrane model (MMM). The aim of the present study was to evaluate the results obtained on the MMM using excised human skin. The amount of ERY penetrating into sebaceous follicles of freshly excised human skin was measured using [N-methyl-14C]erythromycin. The ex vivo penetration of the ion pair ERY/OS into the sebaceous follicles was observed to be doubly enhanced compared with the penetration of the ERY base. The model was shown to be suitable for predicting in vivo penetration of anti-acne formulations into sebaceous glands.