Retinoic acid (RA) induces terminal granulocytic differentiation of the HL-60 promyelocyte leukemia cell line as well as certain other human myeloid leukemias. Specific RA receptors that are members of the steroid-thyroid hormone superfamily of nuclear transcription factors have recently been identified. We developed an HL-60 subclone that was relatively resistant to RA-induced differentiation. Specific nuclear RA receptors in this RA-resistant subclone had a decreased affinity for RA and exhibited a lower molecular weight compared with nuclear RA receptors from the RA-sensitive parental HL-60 cells. Retroviral vector-mediated transduction of a single copy of the RA receptor (RAR-a) into this RA-resistant HL-60 subclone restored the sensitivity of these cells to RA. These observations indicate that RAR-α plays a critical and central role in mediating RA-induced terminal differentiation of HL-60 leukemia cells.