The value of flow cytometric detection of myeloperoxidase (MPO) in the differential diagnosis of acute leukemia was evaluated in 57 cases of acute leukemia and in 9 leukemia cell lines. Cells were fixed and permeabilized with Fix & Perm cell permeabilization kit at room temperature for 15 min each, and stained with anti-MPO monoclonal antibody (MPO-7) by direct immunofluorescence. One myeloid cell line, HL-60, was MPO-positive, while the other myeloid cell lines (KG-1, K-562, and MEG-01) as well as lymphoid cell lines (KM-3, NALM-6, Raji, REH, and T-ALL-1) were MPO-negative as previously described. Among acute leukemias, MPO was detected in 23 of 26 cases of acute myeloid leukemia (AML), 7 of 23 cases of B-lineage acute lymphoblastic leukemia (ALL), 1 of 6 cases of T-lineage ALL (T-ALL), and 1 of 2 cases of acute unclassified leukemia (AUL). The intensity of MPO expression in 6 of 7 B-lineage ALL cases was weak compared with AML labeling. There was no detectable cytochemical MPO in the cells of ALL, AUL, or AML that stained negative for anti-MPO. No relationship between the expression of MPO and myeloid lineage surface antigens was observed in ALL. Three cases of MPO-positive ALL and AUL could be reclassified as biphenotypic leukemia according to the revised Catovsky scoring system. These results indicate that anti-MPO is an excellent marker for the diagnosis and classification of acute leukemia and can be reliably detected by flow cytometry. This rapid technique should be a valuable addition to routine immunophenotyping of acute leukemia.