All-trans-retinoic acid (RA) and 1alpha,25-dihydroxyvitamin D3 (1,25D3) are involved in the control of hematopoiesis and have been suggested to play a role in cellular differentiation and are as such potent inducers of differentiation of myeloid leukemia cells. In this study, we show that, in promyelocytic NB4 cells, addition of 1,25D3 enhances terminal granulocytic RA-dependent differentiation concomitant with the enhanced activation of the RA transcriptional activity through an RARbeta promoter. By EMSA and ChIP assays, we further demonstrate that, while both VDR and RAR are bound to the RARbeta promoter in NB4 cells, addition of 1,25D3 increases VDR binding to this promoter, while that of RA induces the release of VDR and increases the binding of RAR. Thus, contrary to normal myeloid cells, 1,25D3 does not act as a transrepressor of RA transcriptional activity in leukemic cells, suggesting that transcriptional regulation of RA-target genes may be modified in malignant cells. In promyelocytic leukemic cells, the combination of 1,25D3 and RA results in both enhanced transactivation and differentiation.