Differentiation of human promyelocytic leukemia cells, HL-60, has been extensively studied. In this study we utilized 1 alpha,25-dihydroxyvitamin D3 (D3) as a potent inducer and cellular superoxide production as the functional differentiation marker. We examined how nitroblue tetrazolium (NBT) positive cells were produced in the presence of D3. Growth of HL-60 cells tended to level off when the curve was drawn on a logarithmic scale but they grew linearly on a normal scale. When absolute numbers of NBT positive or negative cells were plotted, NBT positive cells only increased linearly in a normal scale, whereas NBT negative cells remained constant after they doubled themselves. When cells were sparsely inoculated in 0.3% agar and cultured for 4 days in the presence of D3, clusters of cells were stained with NBT. Each cluster of cells was composed of one or two NBT negative and three to six NBT positive cells. After treatment with nocodazole and D3, cells were cultured further in the presence of D3. It was demonstrated that only NBT positive cells increased abruptly. Based on these results, differentiated cells might be produced and accumulate through mitosis. In the presence of D3 NBT negative cells remained constant in number and continued to produce NBT positive cells, working as so-called stem cells.