Human whole skin was labeled for 24 h with [6-3H]-glucosamine in organ culture and epidermis, dermis and culture medium were separately analyzed for the molecular mass and content of the [3H]-labeled hyaluronan (HA). Gel filtration on Sephacryl S-1000 of HA purified by HPLC showed a large proportion of the newly synthesized HA to be of a very high molecular mass (greater than 2 X 10(6) Da) in both epidermis and dermis, whereas HA in the medium was of a smaller size. After 24 h chase, most of the high molecular mass HA, and 42-48% of total labeled HA disappeared from both tissue compartments. The size of labeled HA recovered in the chase media was further reduced but the content roughly corresponded to that lost from tissue. The amount of unlabeled HA was not significantly altered in epidermis, whereas in dermis it was reduced to about 10% of the initial values during 5-d culture. The results demonstrate that HA of both epidermis and dermis is synthesized as a very high molecular mass compound but rapidly undergoes a limited degradation into large fragments. The fragmentation of HA is suggested to enhance its diffusion from the tissues, particularly dermis.