Pelage hair follicles were isolated by gentle microdissection from 8-12-day-old rats, and maintained in supplemented Williams E medium. Length measurements made on freshly isolated hair follicles, and at 24-h intervals, showed a significant increase in hair follicle length over 48 h, after which time no further significant increase in length was observed. Photomicrographs of maintained follicles showed that this increase in hair follicle length could be attributed to the production of a keratinized hair shaft. Histology and [methyl-3H] thymidine autoradiography of freshly isolated hair follicles showed the dermal papilla to be elongated, with thymidine uptake located predominantly in the matrix cells of the hair follicle bulb adjacent to the dermal papilla. This pattern remained unaltered for the first 48 h of maintenance, but after 72 h the dermal papilla had rounded into a tight ball of cells, with very little thymidine uptake occurring in the adjacent matrix cells. On maintenance, fetal calf serum (FCS), epidermal growth factor (EGF) and 12-o-tetradecanoyl phorbol 13-acetate (TPA) all significantly stimulated [methyl-3H] thymidine and [U-14C] leucine uptake, but inhibited hair follicle elongation. Insulin-like growth factor-1 (IGF-1) had no significant effect on rates of hair follicle elongation and [methyl-3H] thymidine uptake, but significantly stimulated rates of [U-14C] leucine uptake. Transforming growth factor-beta 1 (TGF-beta 1) significantly inhibited both the rate of [methyl-3H] thymidine uptake and hair follicle elongation.