The main objective of the present work was to investigate the dermal and intracellular delivery of bacitracin, a model polypeptide antibiotic, from ethosomes. Bacitracin and fluorescently labeled bacitracin (FITC-Bac) ethosomes were characterized for shape, lamellarity, fluidity, size distribution and entrapment capacity by scanning electron microscopy (SEM), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), dynamic light scattering (DLS) and ultracentrifugation, respectively. Confocal laser scanning microscopy (CLSM) experiments revealed that ethosomes facilitated the copenetration of antibiotic and phospholipid into cultured 3T3 Swiss albino mice fibroblasts. These results, confirmed by data obtained in fluorescent-activated cell sorting (FACS) experiments, suggest that ethosomes penetrate cellular membrane releasing the entrapped molecule within cells. Additional work was focused on skin permeation behavior of FITC-Bac from ethosomal systems in in vitro and in vivo experiments through human cadaver and rat skin, respectively. These studies demonstrated that the antibiotic peptide was delivered into deep skin layers through intercorneocyte lipid domain of stratum corneum (SC). Occlusion had no effect on the permeation profile of the drug from ethosomes in in vitro experiments. Efficient delivery of antibiotics to deep skin strata from ethosomal applications could be highly beneficial, reducing possible side effects and other drawbacks associated with systemic treatment. Furthermore, ethosomal delivery systems could be considered for the treatment of a number of dermal infections, requiring intracellular delivery of antibiotics, whereby the drug must bypass two barriers: the SC and the cell membrane.