We report a method to establish long-term cultures of melanocytes derived from human hair follicles. Normal human scalp was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by collagenase treatment. Hair-follicle cell suspensions were prepared by trypsin/ethylenediamine tetraacetic acid treatment and cultured in a mixture of Eagle's minimum essential medium (supplemented with 12-O-tetradecanoyl-phorbol-13-acetate and cholera toxin) and keratinocyte serum-free medium. After contaminating fibroblasts and keratinocytes were removed, cells with two distinct morphologies remained. These included large, dendritic and deeply pigmented cells, which did not proliferate and which disappeared by the third passage, and small bipolar cells, which initially were unpigmented, proliferated very rapidly, and became pigmented after the addition of 3-isobutyl-1-methylxanthine to the culture medium. Both cell types were melanocytes as confirmed by electron microscopy and by staining with antibodies to S-100, GD3, and melanosomal antigens. The availability of cultured hair-follicle melanocytes wil facilitate investigations of the role of these cells in normal and abnormal hair biology.