The reactivity of a murine IgG1 monoclonal antibody, NHL-30.5, that detects a surface antigen expressed on acute myeloid leukemia (AML) cells has been studied. Initially raised against the HL-60 cell line, NHL-30.5 has subsequently reacted with blood and/or bone marrow cells from 15 of 19 AML patients studied at presentation or in relapse, 1 patient with chronic myelomonocytic leukemia (CMML), 1 patient with myelofibrosis (MF) who subsequently developed AML and 1 of 5 patients with acute lymphoblastic leukemia (ALL). It has shown no detectable binding to cells from AML patients in remission (0/3), patients with chronic myelogenous leukemia in chronic phase (CML) (0/7), normal bone marrow (0/9), normal peripheral blood mononuclear cells, granulocytes, platelets, erythrocytes, monocytes, or splenocytes by radioimmunoassay or fluorescence analysis using flow cytometry. HL-60 cells induced to differentiate following incubation in the presence of dimethylsulfoxide (DMSO) lost their ability to bind NHL-30.5. Immunoprecipitation of iodinated HL-60 cell surface components showed the antigen to have an apparent MW of 180,000 under reducing conditions. The antigen is apparently different from any other myeloid antigens reported to date and may be useful in further studies of leukemic cell phenotypes.