Formation of the epidermal permeability barrier requires delivery of lamellar body (LB) contents to the stratum corneum interstices. LB are enriched in a mixture of polar lipids and a family of hydrolytic enzymes, required for the extracellular processing of the secreted polar lipids into the more hydrophobic products which mediate barrier function. Prior non-quantitative studies show that acute barrier disruption leads to immediate secretion of the contents of performed LB from the outermost layer of granular cells, followed by the synthesis and accelerated secretion of newly-formed (= nascent) organelles over 0.5-4 h. We asked here whether lipids and hydrolytic enzymes are packaged into nascent organelles separately, or in a parallel, linked process. We first quantified the rate of appearance of lipids (by the content of internal lamellae within LB) and enzyme content (by cytochemistry of neutral lipase and acid sphingomyelinase); both are concentrated in LB, and in nascent organelles. Immediately after barrier disruption, the density of LB in the cytosol of the outermost granular cell decreased by > 50% reduction at 30 min, returning to near-normal densities by 4 h. Nascent organelles budded off a trans-Golgi-like reticulum, in the outermost granular cells as early as 30 min. In quantitative studies, LB progressively accumulated lipid and enzyme contents in parallel. However, when lipid/lamellae generation was inhibited with lipid synthesis inhibitors, enzymes did not accumulate in organelles. Likewise, when exogenous physiologic lipids were delivered to sites of LB generation in the face of brefeldin A blockade of organellogenesis, or when lipids were delivered in conjunction with treatment with lipid synthesis inhibitors, enzymes accumulated only in those organelles that displayed lipid content. These studies demonstrate: (a) quantitative changes in the density of LB in the outermost granular cell at various time points after acute barrier disruption; (b) the origin of nascent organelles in a trans-Golgi-like reticulum; (c) co-ordinate packaging of lipid and enzyme contents into nascent organelles; (d) that lipid deposition in nascent organelles is required for enzyme accumulation; and (e) that enzymes can be delivered to nascent organelles, even if the source of lipid is of exogenous rather than endogenous origin.