Amplification of the c-myc gene in the human promyelocytic leukemia cell line HL-60 is considered to be one of the major causes of its malignant phenotype. It is also well known since the establishment of the cell line that a culture of HL-60 cells contains a small but fixed percentage of spontaneously differentiated cells. We show that the spontaneous differentiation could be a result of extensive losses of amplified c-myc genes by the findings: (a) the spontaneously differentiated HL-60 cells express Mac-1 (CR3, CD11b/CD18) antigen, irreversibly stop the uptake of [3H]thymidine, and die by apoptosis; (b) these cells, when isolated, and when the copy number of c-myc genes is precisely quantitated, show extensive losses of c-myc genes; and (c) low concentrations of hydroxyurea increase the percentage of spontaneously differentiated cells in which the number of c-myc genes is further decreased. A simple theoretical consideration suggests that an active elimination process(es) must be operating besides the stochastic losses of the extrachromosomally amplified c-myc genes by unequal partition at mitosis.