The intercellular lipid sheets of the stratum corneum constitute the epidermal permeability barrier that permits terrestrial life. Although lamellar granules are known to deliver the precursors of the stratum corneum lipids into the intercellular spaces, their site of origin remains unknown. Lamellar granules have characteristics of both secretory granules and lysosomes, which are known to originate from the Golgi apparatus in other cell types. Glucosylceramides, a major component of lamellar granule contents and the precursors of stratum corneum ceramides, have been found to be synthesized primarily in the early compartments of the Golgi apparatus in other cell types. We have investigated the transport and metabolism of a fluorescently labeled ceramide in human keratinocyte cultures using laser-scanning confocal microscopy and lipid analysis. We found that ceramide is metabolized to glucosylceramide and sphingomyelin as it passes through the Golgi apparatus and the metabolites are then delivered to the plasma membrane. Cold temperature, Brefeldin A, and monensin, all known to inhibit transport from the Golgi to the plasma membrane, prevented ceramide metabolites from appearing at the plasma membrane. Because glucosylceramides are one of the most important lipid constituents of lamellar granules, these results support the hypothesis that the Golgi is the origin of lamellar granules.