Long-term cultures of human bone marrow form multinucleated cells (MNC) with many functional characteristics of osteoclasts including: expression of tartrate-resistant acid phosphatase, appropriate responses to osteotropic hormones, calcitonin-induced contraction and formation of resorption lacunae on calcified matrices. However, it is unclear if these cells express similar surface antigens as expressed by authentic osteoclasts, since they form on plastic surfaces in the absence of bone. Bone may be required to complete the differentiation process for osteoclasts. Therefore, we have examined the surface phenotype of MNC and compared it with that of osteoclasts freshly isolated from bone, to determine if MNC express similar surface antigens, and if MNC express antigens which identify their cellular origin. Similar to bone-derived osteoclasts, MNC formed in long-term human bone marrow culture expressed osteoclast-specific antigens (detected by monoclonal antibodies 13c2 and 23c6) and did not express Fc receptors, T cell specific antigens, most myeloid antigens or mature macrophage antigens. In contrast to authentic osteoclasts, MNC reacted with a monoclonal antibody (Mol) which identifies an antigen present on myeloblasts, monocytes, granulocytes, and null cells from human peripheral blood and bone marrow. MNC also reacted with the monoclonal antibody My11, which is present on CFU-GM, the granulocyte-macrophage colony-forming cell, the probable precursor for MNC. These data demonstrate that MNC formed in long-term human marrow cultures express a similar surface phenotype to osteoclasts. This phenotype is different from that expressed by macrophage polykaryons. In addition, MNC also expressed monocyte-related antigens (My11, Mol), suggesting that are derived from or related to the monocytic lineage.