Pig esophageal mucosa has been shown to be a useful and practical substitute for buccal mucosa in in vitro permeability studies in that it offers a larger surface area and it is much easier to prepare. Further, the tissues demonstrate similar histological characteristics. The objectives of this work were to characterize the lipid composition of the esophageal mucosa, to compare it to that of the buccal tissue, and to correlate lipid composition with the membranes' permeability to fentanyl. The major lipid classes of buccal and esophageal epithelia were separated and analysed by automated multiple development high-performance thin-layer chromatography (AMD-HPTLC). The two epithelia presented a very similar lipid pattern. In general, there were more polar lipids than non-polar; glycosylceramides were relatively abundant whereas the amount of ceramides present was very small. The flux of fentanyl applied as the citrate in aqueous solution was comparable across the buccal and esophageal barriers. Lipid extraction provoked a significant increase in permeability. In conclusion, this research confirms the suitability of the esophageal mucosa as a model for buccal permeability studies.