We produced two hybridomas by fusion of mouse myeloma cells with splenocytes from a mouse immunized with the THP-1 human monocytoid leukemia cell line. Two cloned hybridoma cell lines, designated as TM2 and TM3, were obtained. They secreted antibodies against a unique cell surface antigen expressed on all normal peripheral blood monocytes, neutrophilic granulocytes, platelets, and mitogen-induced lymphoblasts, some cells from patients with immature-type lymphoid leukemias. However, the antibodies reacted neither with large numbers of peripheral blood lymphocytes nor with red cells. Cross-blocking studies showed that these monoclonal antibodies recognized the same or a nearly positioned antigen epitope. Immunoprecipitation of THP-1 cell extract with TM2 or TM3 under reducing and nonreducing conditions yielded a specific band of mol wt equal to 120,000 daltons. This determinant appeared to be involved in granulocyte chemotaxis, since neutrophilic granulocytes exposed to TM2 or TM3 showed a significant decrease in chemotaxis toward endotoxin-activated serum. These two monoclonal antibodies did not affect O2- release or luminol-dependent chemiluminescence of neutrophils. Moreover, they did not alter platelet aggregation induced by thrombin. TM2 and TM3 will provide a new reagent in defining the linkage between lymphoid and myeloid differentiation and intermyeloid development.