The cells of the human promyelocytic leukemia cell line (HL60) stop growing and differentiate into macrophage-like cells when exposed to nM concentrations of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). By exposing cells to the frameshift mutagen ICR-191 and subsequently selecting for resistance to the differentiating effects of nM amounts of TPA, we have isolated TPA-insensitive variants. These variants can grow in up to 320 nM TPA concentrations and do not differentiate into morphologically or functionally mature macrophages. The number of phorbol ester receptors, their affinity for phorbol dibutyrate, and the regulation of receptors are the same as for wild-type HL60 cells. As the resistance to TPA increases in the variants, so does the number of cells with increased ploidy. Wild-type HL60 cells are nearly 100% hypodiploid with a modal chromosome number of 43, while a partially TPA-resistant variant (DM30) has 30% hyperdiploid cells with a mean chromosomal number of 70, and a completely resistant variant (DM90) is 93% hyperdiploid averaging 74 chromosomes/cell. The variants differentiate into neutrophils in response to dimethyl sulfoxide but are defective in respiratory burst activity as assayed by the reduction of the dye nitroblue tetrazolium. These variants could be useful in determining the mode of action of TPA in the promotion of tumors.