<i>Aim:</i> The aim of this work was to characterize rabbit ear skin in view of its use in transdermal permeation experiments. <i>Method:</i> The characterization included histological analysis of the tissue, qualitative and quantitative analysis of stratum corneum (SC) lipids, differential scanning calorimetry and permeation experiments (caffeine, nicotinamide, progesterone). As a reference, pig ear skin was used. <i>Results:</i> The results obtained show that rabbit ear skin has a similar SC thickness compared to pig skin although the viable epidermis has a different structure. The lipid composition of rabbit SC was similar to pig SC but was characterized by a lower content of ceramides and a higher content of cholesterol esters and triglycerides. In terms of permeability, rabbit ear skin was 4–7 times less permeable to hydrophilic compounds, probably because of the higher lipophilicity of its SC. The permeability to progesterone was comparable between isolated pig epidermis and rabbit ear skin. <i>Conclusion:</i> Overall, the results obtained in this work support the usefulness of rabbit ear skin as barrier for skin penetration studies, for both lipophilic and hydrophilic permeants.