The composition of internal wool lipids (IWL) resembles that of lipids present in other keratinic tissues such as human hair or the stratum corneum. Advances in the isolation of ceramides from wool and in the characterization of their thermotropic properties could facilitate their application in human skin care treatments. IWL are solvents extracted from wool fibers. Ceramide isolation is carried out by medium-pressure liquid chromatography. The different fractions obtained were analyzed quantitatively by thin layer chromatography coupled to an automated flame ionization detector and by high-performance thin layer chromatography using a densitometric detector. Two important fractions were isolated: one was a mixture of different ceramides and the other was exclusively made of ceramide 2 (nonhydroxy acid sphingosine [NS]). The thermotropic behavior of IWL and their isolated fractions were studied by thermogravimetric analysis, differential scanning calorimetry (DSC), and by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) methodologies. The transition temperature (Tm) obtained was compared with the results of the IWL extract, stratum corneum lipids, and the values found in the bibliography for isolated ceramides. The Tm obtained for IWL (48 degrees C) was lower than that achieved for SCL (65 degrees C). This discrepancy could be due to the different ceramide pattern and to the larger amounts of free fatty acids present in the IWL extract. Although the isolated ceramides had higher Tm values, they resembled the values reported in the bibliography. The suitability of the fraction composed exclusively of ceramide 2 [NS] for skin applications was confirmed by ATR-FTIR and DSC.