Ascorbate has been related to the differentiation of several mesenchymal cells including haematopoietic cells. We have previously demonstrated that ascorbate enhances the activity of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) on monocytic differentiation of HL-60 cells. Here, we show that ascorbate-mediated modification of cellular redox state and AP-1 (activating protein-1) DNA binding during early phases are related to the enhancing effect of ascorbate on differentiation. Ascorbate, but not its fully oxidized form, dehydroascorbate, or an ascorbate analogue with a low rate of oxidation, ascorbate-2-phosphate, enhanced the differentiation induced by 1 alpha,25(OH)2D3, modified cytosolic reactive oxygen species levels and mitochondrial redox potential (delta psi m), and modulated AP-1 DNA binding in HL-60 cells. Ascorbate itself increased AP-1 binding to DNA in noninduced cells, whereas it inhibited AP-1 binding in 1 alpha,25(OH)2D3-induced cells. However, ascorbate increased the mRNA levels of c-jun, junB, and c-fos in 1 alpha,25(OH)2D3-induced cells. Taken together, these results suggest that the enhancing effect of ascorbate on HL-60 differentiation induced by 1 alpha, 25(OH)2D3 is related to its effect on the cellular redox state and the modulation of AP-1 activity.