Leukemic cells from a child with acute lymphoblastic leukemia (ALL) had high natural killer (NK) activity against K562 as determined by the 51Cr release assay at a 40:1 effector:target ratio: percent lysis was 76.8% (147.4% of normal lymphocyte value) and higher than that of control leukemic cells from 12 childhood ALL (0.1% +/- 0.3%). Two leukemic cell lines (SPI-801 and SPI-802) were established from the patient, and they were essentially the same as the freshly harvested leukemic cells in their morphology, cytochemistry, immunologic markers, and functions. The cultured cell lines as well as the fresh leukemic cells had receptors for sheep red blood cells, IgG-Fc, and C3. The cultured cells were OKM1+, Ia+ and asialo-GM1+, and were OKT-3-, OKT-4- , OKT-6-, OKT-8-, Leu-7-, human monocyte-, common ALL-, T cell-, surface Ig-, and cytoplasmic IgM- as determined by the immunofluorescence method. The two cell lines shared the same chromosome abnormalities. Their chromosomes were in hypotriploid region (63--73), and 6q-, 11p+, and several marker chromosomes were demonstrated. They had spontaneous cytotoxicity against K562 (percent lysis: up to 17.8% and 33.5% of normal lymphocyte value in SPI-801 and SPI-802, respectively) and Molt-3 (38.2% and 27.8% of normal lymphocyte value), but not against Raji and mitogen-induced normal lymphoblasts. Such phenotypic and functional characteristics of the fresh leukemic cells and cultured cells are virtually identical to those of NK cells, demonstrating a new phenotype of childhood ALL of NK cell origin.