We have made a new monoclonal antibody, EL-2, and used it with an immunorosetting procedure combined with Ficoll-Hypaque gradient centrifugation to purify and culture basal keratinocytes. Immunofluorescence of cell suspensions and immunoperoxidase staining of tissue sections demonstrate that EL-2 reacts with malignant cell lines, activated lymphocytes and monocytes, and basal keratinocytes. Sequential immunoprecipitation studies demonstrate that monoclonal antibodies EL-2 and 4F2 detect the same membrane protein. However, we have extended previous studies by making the new observation that both EL-2 and 4F2 react with cultured melanocytes. Basal keratinocytes were purified from single-cell epidermal suspensions by incubation with EL-2 followed by rosetting with rabbit antimouse IgG antibodies covalently linked to bovine red blood cells. Rosetting (basal) keratinocytes were separated from EL-2 negative cells by Ficoll gradient centrifugation. We obtained basal keratinocyte populations of greater than 90% purity as assessed by reactivity with EL-2 and another basal keratinocyte-specific monoclonal antibody, HCl. Langerhans cell, fibroblast, and melanocyte contamination was negligible. Cultures of basal keratinocytes were enriched in EL-2-reactive cells throughout the entire 19 days of culture studied. EL-2 is being used to characterize disorders of keratinocyte proliferation; EL-2 reacted with both squamous and basal cell carcinomas. EL-2 stained only the basal layer of lesional skin from patients with psoriasis, pityriasis rubra pilaris, and Darier's disease. Purification of basal keratinocytes will be important in biochemical and functional studies of normal skin and in establishing long-term keratinocyte lines from normal cells.