Either all-trans-retinoic acid (RA) or vitamin D3 (VD) induces differentiation of the myeloid leukemia cell line HL-60. RA is available for the treatment of acute promyeloleukemia, although the development of resistance to the agent is a serious problem for differentiation-inducing therapy. To approach the mechanisms of resistance to RA, we developed two novel cell lines, HL-60-R2 and R9, which were subcloned after exposure to increasing concentrations of RA. The growth rate of HL-60-R2 cells was significantly increased by RA treatment, whereas the growth rate of HL-60-R9 was not affected. RA induces apoptosis in the parental HL-60 cells. The number of apoptotic cells, however, was not increased and nitroblue tetrazolium (NBT) reduction was not altered by 1 microM RA in either of the cloned cell lines. Treatment with VD induced monocytic differentiation and increased the expression of CD11b in HL-60 and HL-60-R9 cells, but not in HL-60-R2 cells. Flow cytometric and G-banding analysis demonstrated that R2 cells were near-triploid. The sequencing analysis revealed a deletion of three nucleotides in the sequence of the RAR alpha gene in HL-60-R9 cells, resulting in deletion of codon 286. No mutation was found in HL-60-R2 cells. Taken together, these data indicate that the resistance to RA is caused by the mutation in RAR alpha of HL-60-R9, but by other factor(s), which also affect the VD-response pathways, in HL-60-R2. The abnormal response to VD may be associated with the abnormal ploidy of the R2 cells.