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Abundant hyaluronan is present between epidermal keratinocytes. However, virtually nothing is known regarding its organization in the limited extracellular space between these cells. We have used metabolic labeling with [³H]glucosamine and [³⁵S]sulfate and a hyaluronan-specific biotinylated probe to study the metabolism of hyaluronan and its localization in monolayer cultures of a rat epidermal keratinocyte cell line. Hyaluronan (∼20 fg/cell) was present on the apical and lateral surfaces of the cells in two nearly equal pools, either in patches (∼160/cell) or diffusely spread. The hyaluronan in the patches is bound to CD44 as indicated by co-localization with an antibody to CD44, and by displacement with hyaluronan decasaccharides as well as with an antibody that blocks hyaluronan binding to CD44. The inability of hyaluronan oligomers shorter than 10 monosaccharides to displace hyaluronan suggests that …