Atopic dermatitis is a chronic remittent skin disease. In the extrinsic form of atopic dermatitis, type IgE-mediated reactions play an important pathophysiological role. The aim of the present study was to examine whether type I allergens can penetrate into the skin. Therefore, pollen proteins were labeled with fluorescein isothiocyanate (FITC), and their penetration profile was studied qualitatively. Solutions of FITC-labeled pollen proteins were applied in vitro on porcine skin and in vivo on human skin. In vitro, the FITC-labeled proteins were observed within the complete stratum corneum (SC) and inside the hair follicles even 15 min after application. They were also distributed inside the dermis around the hair follicles. In vivo, a similar pattern of distribution within the SC and the hair follicles was observed. These results indicate penetration via the SC lipid layers and a faster penetration via the hair follicles. The FITC-labeled proteins entered the dermis via the follicular pathway. Therefore, the follicular penetration should be considered in the development of skin protection strategies. To evaluate such strategies, the developed method can be used, and further studies in atopic dermatitis patients are necessary to determine whether the penetration of type I allergens is increased.