Hyaluronan has recently been introduced as a vehicle for topical application of drugs to the skin. We sought to determine whether hyaluronan acts solely as a hydrophilic reservoir on the surface of intact skin or might partly penetrate it. Drug-free hyaluronan gels were applied to the intact skin of hairless mice and human forearm in situ, with and without [3H] hyaluronan. [3H]hyaluronan was shown by autoradiography to disseminate through all layers of intact skin in mouse and human, reaching the dermis within 30 min of application in mice. Cellular uptake of [3H]hyaluronan was observed in the deeper layers of epidermis, dermis, and in lymphatic endothelium. Absorption through skin was confirmed in mice by chromatographic analysis of blood, urine, and extracts from skin and liver, which identified 3H as intact hyaluronan and its metabolites, free acetate and water. Hyaluronan absorption was similarly demonstrated without polyethylene glycol, which is usually included in the topical formulation. [3H]hyaluronan absorption was not restricted to its smaller polymers as demonstrated by the recovery of polymers of (360-400 kDa) from both blood and skin. This finding suggests that its passage through epidermis does not rely on passive diffusion but may be facilitated by active transport. This study establishes that hyaluronan is absorbed from the surface of the skin and passes rapidly through epidermis, which may allow associated drugs to be carried in relatively high concentration at least as far as the deeper layers of the dermis.