The surface of dendritic cells from mouse spleen, thymus, and epidermis has been compared with a panel of monoclonal antibodies and the FACS. A method was first developed to isolate populations of large, adherent, thymic dendritic cells that were greater than 90% pure. These were released by collagenase digestion and separated from adherent macrophages after overnight culture. Enrichment was based on the facts that most macrophages remained plastic adherent and rosetted strongly with antibody-coated erythrocytes. As in spleen, thymic dendritic cells were stellate in shape, had abundant class I and II MHC products, lacked many standard macrophage and lymphocyte markers, and actively stimulated the mixed leukocyte reaction. Most spleen and thymic dendritic cells could be lysed by the 7D4 mAb, to the low-affinity IL-2 receptor, and complement but the levels of 7D4 by FACS were low and sometimes not above background. Differences among dendritic cells from different tissues were noted with other mAb. Adherent dendritic cells from thymus all expressed the J11d "B cell" antigen and the NL145 interdigitating cell marker, but lacked the 33D1 spleen dendritic cell antigen. Eighty to ninety percent of spleen dendritic cells were J11d-, NL145-, 33D1+ but the remainder expressed the J11d+, NL145+, 33D1- thymic phenotype. The latter phenotype also was identical to that of epidermal Langerhans' cells. We postulate that the major 33D1+ cell in spleen represents a migratory stage in which dendritic cells are moving from tissues to lymphoid organs.