Two distinct differentiation antigens of human myelomonocytic cells are defined using murine monoclonal antibodies. The antigens recognized by antibodies 20.2 and 20.3 are expressed by all cells of the monocyte lineage in both peripheral blood and bone marrow. Cell-sorting experiments demonstrated that histiocytes and immature bone marrow cells with detectable alpha-naphthyl butyrate esterase activity also express both antigens. Within cells of other lineages, the antigens had distinct patterns of expression. Immature myeloid cells were 20.2 negative, but 20.3 positive; whereas mature myeloid cells were 20.2 positive, but 20.3 negative. Nucleated erythroid cells and platelets expressed only the 20.3 antigen. These results indicate that myeloid and monocytic cells share common differentiation antigens with cells of the erythroid and megakaryocytic lineages. The 20.2 and 20.3 antibodies reacted with the leukemic cells from some patients with acute nonlymphocytic leukemia (FAB, M1-M5) and with some cell lines derived from patients with nonlymphocytic leukemia, but not with blast cells from patients with lymphoid leukemia or with lymphoid leukemic cell lines. These antibodies may prove useful in studying the differentiation of bone marrow stem cells, in defining the cellular origins and classification of leukemias, and in the identification of distinct prognostic subgroups of acute nonlymphocytic leukemia.