- The aim of this study was to explore the transdermal delivery potential of a new caffeine-containing microemulsion system. The skin permeability of caffeine (CAF) was measured in vitro using skin excised from three different animal species: rat, rabbit and pig. As shown, microemulsion containing 20% aqueous phase enhanced CAF permeation across fresh rat skin by one order of magnitude ( P app = 8.2 × 10 −3 vs. 0.86 × 10 −3 cm/h; enhancement ratio = 9.6). The permeability coefficient value, the cumulative permeation amount, and the percent of dose permeated after 24 h, decreased with the increase of water content from 60% to 80% in microemulsions due to the apparent increase in the droplet size. Importantly, differences were noted between caffeine transport rates across fresh and frozen/thawed pig skin whereas microemulsions delivered caffeine at similar rates across rat and rabbit skin, either fresh or frozen/thawed. It has been shown that the permeability of caffeine through frozen/thawed pig skin was abnormally high and was independent of its vehicle properties, i.e., its hydrophilic or lipophilic nature. It has been hypothesized that the reason for this abnormality is that porcine stratum corneum has a higher ceramide-to-cholesterol ratio compared to rat and rabbit skin. This unusual phenomenon observed in a non-freshly used porcine skin places a question mark on its suitability to in vitro evaluation of transdermal drug delivery systems.