A new myelomonoblastic cell line (M20) was established from the peripheral blood of a ten-year-old child with acute myeloblastic leukemia, using an improved method for supporting the initial stages of cell proliferation. The addition of irradiated macrophage monolayers to the proliferating cells appeared to overcome the deterioration of the primary cultures and enable them to continue proliferating until they became independent of this environment. The cell line that developed consisted of myeloblasts and promyelocytes characterized by light and scanning electron microscopy, cytochemistry, and enzymatic activities. The cells expressed Fc receptors and WT1 antigens but did not exhibit HLA-DR, HMA1, Epstein-Barr virus nuclear antigen, and surface Ig. The M20 cells produced colonies when cultured in semisolid medium and secreted lysozyme, prostaglandin E2, and interleukin 1. An attempt was also made to analyse the position of the M20 cells in the scheme of differentiation of the myelomonocytic lineage using different approaches. Treatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate induced their adherence to plastic surfaces and partial maturation to macrophages as judged by morphological criteria, cytochemistry, and enzyme activities. However, comparison of the M20 cells to other well-established myelomonoblastic cell lines did not reveal any pattern suggesting a possible relationship between surface markers, cell function, and differentiation pathway of the various cell lines tested. Establishment of additional cell lines and identification of new markers may assist in defining the mechanisms involved in normal differentiation and malignant transformation of this cell lineage. In addition, such cell lines may also provide a tool for the quantitative recovery of a variety of monokines.