The microstructure formed by intercellular lipids in the stratum corneum is important for the barrier function of the skin. However, the correlation between lipid composition and microstructure has not yet been clarified. To elucidate the microstructure of intercellular lipids in the stratum corneum, an intercellular lipid model was prepared from ceramide 5 (CER5), cholesterol (CHOL), and palmitic acid (PA), considering the nonuniformity of the lipid components of the stratum corneum. A response surface method incorporating thin-plate spline interpolation (RSM-S) was employed to prepare the CER5/CHOL/PA lipid bilayers. Fluorescence anisotropy of the CER5/CHOL/PA bilayers showed four distinct clusters based on Kohonen's self-organizing maps (SOM). At the centroid formulation of those clusters, the microstructures of CER5/CHOL/PA bilayers were determined using synchrotron X-ray scattering. Three kinds of lamellar structures and two kinds of lateral packing-namely, hexagonal and orthorhombic-were formed. The microstructure of the CER5/CHOL/PA bilayers was likely to be intrinsic to the intercellular lipids in the stratum corneum. In conclusion, the CER5/CHOL/PA bilayers prepared based on RSM-S and SOM were useful as models of the intercellular lipids in the stratum corneum.