The expression of hyaluronan (HA) in fetal human skin was studied by using a biotinylated HA-binding probe. The uniform expression of HA in primitive skin was changed after the 9th week, when differentiation of the basement membrane zone increased HA in the subepidermal mesenchyme. Maturation of the papillary dermis at the 12-20th weeks led to the thickening of this HA-enriched zone; the underlying reticular layer was less intensely stained. In epidermis the number of cell layers rapidly increased after the 9th week. At first all epidermal layers were HA-positive. A complete loss of HA from the upper intermediate cells on the 18th week preceded the formation of mature granular and cornified layers. Peridermal cells remained HA-positive even when the underlying stratum corneum turned negative. The tightly apposed basal epithelial cells, the first stage of hair follicle and eccrine sweat gland formation, became almost completely depleted of HA. With advancing bulb development HA returned in the epithelial compartment, until maturation of the hair follicles restricted its expression to the outer root sheath and hair matrix. Maturation of the sebaceous glands led to the expression of HA pericellularly in the germinative cells and intracellularly in the mature sebocytes. Marked changes thus occur in the distribution of HA during fetal skin development; the primitive tissues exhibited a uniform widespread expression of HA, and maturing tissues showed distinct locally regulated patterns. The loss of epithelial HA in the hair follicle anlagen and upper intermediate cells turned out to be early differentiation markers.