Recently, hair follicles were shown to be of great importance concerning the penetration and reservoir behavior of topically applied substances. Especially microparticles are known to be efficient drug carriers into the hair follicles. It has been shown in various investigations that, depending on their size, microparticles can be utilized to enhance the penetration of substances into the hair follicles. In the present study, it was investigated whether they could also be used to block the hair follicles to stop penetration. For this purpose, the follicular penetration of a sodium fluorescein-containing hydrogel and follicular reservoir depletion were investigated by means of differential stripping in vivo. In a second study design, the skin areas were pretreated with microparticles 5 µm in diameter. Subsequently, the follicular penetration of sodium fluorescein-containing hydrogel was likewise investigated by differential stripping. The results showed that the hair follicle infundibula contained a significant fluorescent signal when sodium fluorescein in hydrogel was applied. In contrast, in the case of pretreatment with microparticles, only a very low fluorescence signal was measurable in the hair follicle infundibula. The microparticles clearly blocked the follicular orifices so that a penetration of sodium fluorescein was almost completely inhibited. This was confirmed by histological investigations. Thus, depending on their size, microparticles act as efficient drug carriers or can be utilized as follicle blockers to stop the penetration of topically applied substances. This could be very useful in skin protection to prevent the storage of harmful topically applied substances in the hair follicles, known to represent long-term reservoirs.