This study aimed to evaluate the penetration of Methylene Blue (MB)-loaded microspheres into the follicular ducts of rats. MB was incorporated into porous nylon microspheres which have a size of 10 ?m (Orgasol 4000 Nat Cos, Elf-Atochem, France). The microspheres were dispersed into fluid silicone. Male hairless rats (NU, Iffa Credo, France; mutant hairless Sprague Dawley rat) were used to evaluate the penetration of MB into hair follicles. After formulation application, MB diffusion was induced and skin biopsies were realized immediately, 2 hours and 26 hours after MB loaded microspheres application. All samples were snap frozen in liquid nitrogen and processed stored at -80°C before embedded in TissuTek and cryostat-cut. 14?m sections were realized. MB fluorescence was observed with Eclipse E800 microscope (Nikon, Tokyo, Japan) expanded for fluorescence microscopy (with FITC, TRITC and CY5 filters) and penetration depth was quantified using image analysis software (Sigma Pro, Jandel, USA). Autofluorescence was evaluated using FITC and TRITC filter. MB was seen 200?m deep in hair follicles (2 hours after application) and at 600?m (26 hours after application). This galenical approach aims to transport MB into the hair follicle specifically and deeply.