A wide range of different agents are capable of inducing the onset of HL-60 differentiation along the myeloid lineage. The diversity of these agents has made the analysis of the molecular mechanisms involved in the regulation of the onset and progress of terminal differentiation in these cells difficult. We have adapted the standard soft-agar cloning procedure to enable the single-step selection of clonal populations of spontaneously arising differentiation resistant HL-60 variants. This simple procedure which obviates the need for mutagenesis, long-term exposure to inducing agents or complex manipulations, optimises the chance of obtaining variants with a single lesion blocking the onset of differentiation. The analysis of two variants, HL-R5 and HL-D4, selected by this procedure for resistance to retinoic acid and DMSO, respectively, suggests the existence of different pathways used by the two agents which converge before the onset of terminal differentiation.