With the use of model systems, previous work has identified the relative positions of several of the lipid subclasses (17)(18)(19). CERs with a nonhydroxyl C24 acyl chain linked to a C18 sphingosine (NS) and FFAs were located in the central region of the LPP trilayer structure, while CHOL remained exclusively in the outer region (18). CER EOS is uniquely positioned in both the outer and interior regions, with the head groups present at the boundary of the unit cell, while the carbon chain extends to the central head Abstract The lipid matrix of the stratum corneum, the outermost skin layer, consists primarily of ceramides, cholesterol, and FFAs. These lipids form a trilayer long-periodicity phase (LPP) that is unique to this barrier. Knowledge about the LPP is essential in understanding the barrier function. Previous studies of LPP lipid models have identified the position of the major lipid classes and suggested that a large fraction of FFAs and the ceramide acyl chain are present in the central region. However, the precise arrangement, such as lipid subclass mixing (isolated or mixed) and ceramide conformation (extended or hairpin), remains unknown. Here, we deuterated FFAs and the ceramide acyl chain to study CD 2 and CH 2 interactions with Fourier-transform infrared spectroscopy. The ceramide and FFAs of various chain lengths were not in separate domains but had mixed together. The larger number of CD 2 -CD 2 lipid chain interactions in the LPP than in a symmetrical bilayer structure implied that the ceramide had primarily adopted an extended conformation. Shorter FFAs were present in the central region of the LPP. This model explores the biophysical properties of the stratum corneum's LPP to improve the understanding of the barrier function of this layer.-Beddoes, C. M., G. S. Gooris, and J. A. Bouwstra. Preferential arrangement of lipids in the long-periodicity phase of a stratum corneum matrix model.