Granulocytic-macrophage colony in vitro formation depends on the presence of the colony stimulating factor (CSF). When, in double layer agar method, the CSF is provided from leucocyte underlayers, the pre-incubation time of the feeder-layers must be kept constant. The results obtained in this study have demonstrated in fact that the growth of the colonies can be remarkably different if feeder-layers are incubated for 1, 7 or 14 days.