The lipid regions in the outermost layer of the skin (stratum corneum) form the main barrier for diffusion of substances through the skin. In this layer the main lipid classes are ceramides, cholesterol (CHOL), and FFA. Previous studies revealed a coexistence of two crystalline lamellar phases with periodicities of approximately 13 nm (referred to as long periodicity phase) and 6 nm (short periodicity phase). Additional studies showed that lipid mixtures prepared with isolated pig ceramides (pigCER) mimic lipid phase behavior in stratum corneum closely. Because the molecular structure of pigCER differs in some important aspects from that of human ceramides (HCER), in the present study the phase behavior of mixtures prepared with HCER has been examined. Phase behavior studies of mixtures based on HCER revealed that in CHOL:HCER mixtures the long periodicity phase dominates. In the absence of HCER1 the short periodicity phase is dominant. Addition of FFA promotes the formation of the short periodicity phase and induces a transition from a hexagonal sublattice to an orthorhombic sublattice. Furthermore, the presence of FFA promotes the formation of a liquid phase. Finally, cholesterol sulfate, a minor but important lipid in the stratum corneum, reduces the amount of cholesterol that phase separates in crystalline domains. From these observations it can be concluded that the phase behavior of mixtures prepared from HCER differs in some important aspects from that prepared from pigCER. The most prevalent differences are the following: i) the addition of FFA promotes the formation of the short periodicity phase; and ii) liquid lateral packing is obviously present in CHOL:HCER:FFA mixtures. These changes in phase behavior might be due to a larger amount of linoleic acid moiety in HCER mixtures compared with that in pigCER mixtures.