The mammalian epidermal permeability barrier is provided by highly hydrophobic lipids forming multiple membrane bilayers within the extracellular domains of the outer, cornified cell layers. To characterize the critical events associated with barrier maturation, we correlated the emergence of a competent barrier to transepidermal water loss with development of the lamellar body secretory system, the organization of stratum corneum membrane bilayers, and the lipid composition of these membranes in the perinatal rat. Whereas pups of 19 d estimated gestational age had no measurable barrier (transepidermal water loss greater than 10 mg/cm2/h), by 21 d the barrier was well established (mean transepidermal water loss 0.41 mg/cm2/h). Development of a functional barrier correlated with increasing thickness of the stratum corneum, as well as with development of a membrane pattern of lipid deposition, visualized with the hydrophobic fluorescent probe nile red. At 19 d estimated gestational age, the stratum corneum intercellular domains exhibited an abundance of secreted lamellar body contents, but they were not organized into basic bilayer unit structures. Lamellar unit structures became evident by 20 d and extended throughout the stratum corneum interstices by 22 d (term). The quantity of lipid in isolated stratum corneum increased significantly between 19 and 20 d (34.08 versus 50.08 mean micrograms lipid/cm2, respectively; p less than 0.02) and still further between 20 and 21 d estimated gestational age (74.49 micrograms lipid/cm2; p less than 0.001). This increase was due to progressive accumulation of neutral lipids, particularly cholesterol, as well as nonpolar ceramides, as shown by thin-layer chromatography/scanning densitometry. These studies imply that in the development of cutaneous barrier function in the fetal rat both the generation of sufficient quantities of hydrophobic lipids and the organization of these lipids into bilayer unit structures are required.