The steroid hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induces maturation of many cells, including HL-60 promyelocytic leukemia cells that are differentiated to monocytes/macrophages. This process involves changes in transcription of genes such as c-myc. We investigated the effects of 1,25(OH)2D3 on nuclear protein binding to the c-myc intron element (MIE), a region of DNA within the c-myc gene. A mutation in this MIE sequence has been shown to be associated with uncontrolled expression of the c-myc gene in various cell lines. In this report, we demonstrate for the first time that 1,25-(OH)2D3 induces increased binding of nuclear proteins to this MIE in HL-60 cells. The major MIE-binding proteins were approximately 32 kDa in size. Interestingly, phorbol 12-myristate 13-acetate induced a similar increase in binding of the 32-kDa doublet protein to the MIE. In addition, we showed that the level of 138-kDa MIE-binding protein was increased by 1,25- (OH)2D3 and phorbol 12-myristate 13-acetate. However, the extent of MIE binding by the 138-kDa protein is significantly less than that of the 32-kDa doublet binding species, MIE binding by the 32-kDa doublet protein was significantly increased within 12 h of 1,25(OH)2D3 treatment. The time course of this increase was similar to that of 1,25-(OH)2D3-induced inhibition of c-myc gene transcription. We also demonstrated that dephosphorylation of the 32-kDa doublet protein inhibited its binding to the MIE. Thus, this study shows that the mechanism employed by 1,25-(OH)2D3 for regulation of c-myc expression may involve an increase in protein binding to the MIE, which has been shown to be the site for control of c-myc gene expression.