Despite a number of studies on the composition of the lipids of stratum corneum (SC) and sebum, questions remain about the detailed molecular arrangement of the two superficial components of human skin. The investigation of the molecular components of SC <i>in vivo</i> is important to understand the function of what was once thought to be a "dead" epithelium. We have investigated the molecular composition of SC and sebum <i>in vivo</i>, in the mid-infrared, with fiber-based attenuated total reflection Fourier transform infrared spectroscopy (ATR/FT-IR). This technique combines the sensitivity of infrared spectroscopy in detecting molecular composition and conformational order with the capability of probing surfaces to a depth of less than 1 μm. ATR/FT-IR is therefore particularly useful for the investigation of interfaces such as SC and the sebaceous layers. We found that with the use of ATR/FT-IR one can distinguish between the contribution of the molecular components of sebum and SC. The presence of spectral "signatures" of the lipids of sebum allowed us to improve the interpretation of some infrared bands of sebaceous origin as well as of SC <i>in vivo</i>. We also found that ATR/FT-IR can be used to separate the spectral contributions of sebum and SC, and as a method to study the early recovery of superficial lipids after the removal of sebum. Following calibration, a method can be developed to quantify the relative amount of fatty acid in sebum with the use of ATR/FT-IR. We observed that the sebaceous fatty acids that reach the surface of the skin recover at a slower rate than other sebaceous lipids. Our investigation shows that fiber-based ATR/FT-IR is a promising spectroscopic approach to the study of epithelial surfaces and surface contaminants <i>in vivo</i>.

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