Induction of differentiation of human promyelocytic leukemia cell line HL-60 by dimethyl sulfoxide (Me2SO) was analyzed to determine the relationship between exposure time of the inducer and cell cycle. A minimum incubation time of 12 hr with Me2SO was required in order to induce differentiation in a small but significant proportion of cells. These expressed differentiation markers (morphology, phagocytosis, and nitroblue tetrazolium reduction) up to 12 hr after Me2SO was removed from the medium. For periods beyond 12 hr and as long as 120 hr of contact of HL-60 cells with the inducing agent, a linear rise in the percentage of differentiated cells was observed. The sensitivity to Me2SO of HL-60 cells synchronized by double thymidine block was examined and was found to be similar to that of nonsynchronized cells. The effect of Me2SO was not altered when incubated with cells at different phases of the cell cycle. Finally, even nonproliferating cells were sensitive to the inducing effect of Me2SO. The data are consistent with a stochastic model of induction to differentiation without having any linkage to the cell cycle.