To establish if epidermal Langerhans cells (LC) are related to spleen dendritic cells, we have considered the morphology, phenotype, and function of the 2 cell types in culture. Cultured LC could be partially enriched (up to 50%) on the basis of 2 simple physical properties: nonadherence to plastic, and low buoyant density in dense albumin columns. The morphology of cultured LC and spleen dendritic cells were similar. In particular both cell types had many cell processes and/or veils, and cultured LC lost their distinguishing Birbeck granules. Freshly isolated LC exhibited nonspecific esterase and ATPase, as well as the F4/80 (alpha-macrophage) and 2.4G2 (alpha-Fc receptor) antigens. However all these traits were lost in culture, while Ia and Mac-1 antigens persisted. As a result, the cytochemical and antigenic phenotype of LC became similar to spleen dendritic cells. The one exception was that LC lacked the 33D1 dendritic cell antigen. The function of LC at first differed from spleen dendritic cells in that fresh LC were weak stimulators of T cell proliferation in the mixed leukocyte reaction and in sodium periodate-induced mitogenesis. However, stimulatory activity per cell increased at least 30 fold in culture so that by 2-3 days, LC were 3-10 times more potent than dendritic cells. Maturation of LC function was radioresistant and was accompanied by a small increase in cell surface Ia antigens. Although LC have been likened both to lymphoid dendritic cells and to macrophages, our data suggest a different conclusion. LC seem to be dendritic cell precursors and are immunologically immature. Possibly, lymphoid dendritic cells are in general derived from substantial pools of precursors in nonlymphoid tissues, such as epidermal LC.