Liposomes had been widely used for drug delivery in the past. In this study, five different liposomes were used as a follicular delivery system in pig ear skin. The liposomes mainly differed in their sphere diameter, lipid composition, and surface charge. A novel class of liposomes being amphoteric in their charge behavior are compared to established anionic and cationic liposomes. Two different fluorescent dyes, hydrophilic carboxyfluoresceine or lipophilic curcumin, were enclosed in the liposomes and used as model drugs. The fluorescent dyes were also applied in a standard formulation for reference. The penetration depth of the dyes was measured by laser scanning microscopy in histological sections. One hour, 3, 5, and 7 days after application, biopsies were taken and the penetration depth into the hair follicle was measured in longitudinal sections. The liposomes showed a higher penetration depth compared to the standard formulation. The relative penetration depth of the dyes, applied in the standard formulation, averaged 30% of the full follicle length during the whole observation period, whereas the liposomal formulations penetrated considerably deeper into the hair follicles. Amphoteric and cationic liposomes reached an average relative penetration depth of approximately 70% of the full hair follicle length.