Background: Flow cytometry may be used to determine immunophenotype or lineage of leukemic cells, but few antibodies are available that are specific for cells of monocytic and granulocytic lineage. Objective: The purpose of this study was to evaluate the flow cytometric staining patterns of 3 commercial monoclonal antibodies for monocytes and granulocytes in clinically healthy dogs and in dogs with acute myeloid leukemia (AML). Methods: Mouse antihuman macrophage antibody (MAC387), mouse anti-human myeloperoxidase (MPO), and a canine neutrophil–specific antibody (NSA) were evaluated using flow cytometry on blood from 6 clinically healthy control dogs, and on blood (n = 7) and/or bone marrow (n = 2) from 8 dogs with AML. A diagnosis of acute leukemia was confirmed by >30% blasts in bone marrow or >30% blasts in peripheral blood, together with bi- or pancytopenia, circulating CD34-positive blast cells, and clinical signs of disease. Leukemic samples also were evaluated using a wide panel of monoclonal antibodies. Results: MAC387 stained neutrophils and monocytes from control dogs, although the staining profiles for the 2 cell types differed. MPO and NSA resulted in strong positive staining of neutrophils; MPO also stained monocytes weakly. Lymphocytes did not stain with any of the antibodies. One case was classified as AML of granulocytic lineage (AML-M1), 6 cases were classified as acute monocytic leukemia (AML-M5), and 1 case was classified as acute myelomonocytic leukemia (AML-M4). Neoplastic myeloblasts in the dog with granulocytic AML were positive for MPO, NSA, MAC387, and CD4. All monoblasts from the dogs with AML-M5 were positive for CD14, 5 of 6 were positive for MAC387, and 2 were positive for MPO. NSA staining was negative in the 2 dogs with AML-M5 in which it was evaluated. In the dog with AML-M4 variable percentages of blast cells were positive for CD14, MPO, MAC387, CD4, and NSA. Conclusions: Antigens identified by antibodies to MAC387, MPO, and NSA were expressed not just by normal mature neutrophils and monocytes, but also by neoplastic myeloblasts and monoblasts. These 3 antibodies may be useful as part of a wider panel for immunophenotyping AML in dogs.