We have studied the interactions of dimethyl sulfoxide (DMSO), Giant Cell Tumor (GCT) cell‐conditioned medium (GCT CM), and highly purified granulocyte‐macrophage colony‐stimulating factors (GM‐CSF) on the growth and maturation of a highly passaged population of HL‐60 cells. DMSO produced dose‐dependent inhibition of HL‐60 growth in liquid and semisolid media. Growth was partially to completely restored by the addition of GCT CM to cultures. Experiments in which cell volume, cell cycle kinetics, tritiated thymidine (3HTdr) incorporation, cell number, and nitroblue tetrazolium (NBT) reduction were compared during culture indicated that DMSO inhibited the spontaneous increase in cell volume and flow of cells through the cell cycle which occurred in the first day of culture, the increase in 3HTdr incorporation which was detectable by day 2; and the increment in cell counts which occurred by day 3. These effects were opposed by GCT CM. In contrast, the DMSO‐induced increase in NBT reduction which occurred by day 6 was not influenced by GCT CM. The major principle opposing DMSO was GM‐CSF, since (1) highly purified GM‐CSF from GCT cells and recombinant GM‐CSF from COS cells transfected with the Mo cell GM‐CSF gene overcame greater than 50% of DMSO inhibition; and (2) conditioned media from cells not producing CSF, G‐CSF from GCT cells, and recombinant G‐CSF from Escherichia coli transfected with the G‐CSF gene from 5,637 cells were inactive. DMSO had little or no effect on the elaboration of autostimulatory activity by HL‐60 cells. DMSO is a useful agent for inhibiting the spontaneous growth of HL‐60 cells and restoring their dependence on GM‐CSF, a property which may be mediated through the effects of DMSO on cell cycle kinetics and/or maturation.