Studies were performed to determine the regulation of DMT1 (divalent metal transporter 1) during RA (retinoic acid)-induced differentiation of P19 embryonic carcinoma cells. Protein and mRNA expression for the +/-IRE (iron response element) forms of DMT1, but not the 1A isoform, were down-regulated within the first few hours upon removal of RA, at which time the cells began to differentiate. The turnover of the +/-IRE isoforms of DMT1 protein during this period was found to be dependent on both the proteasomal and lysosomal pathways. Changes in mRNA levels were shown to be regulated by nitric oxide produced by the induction of neuronal nitric oxide synthase after removal of RA. Nitric oxide functions by inhibiting NF-kappaB (nuclear factor kappaB) nuclear translocation and the subsequent binding to the putative NF-kappaB response element (at -19 to -23) within the 1B promoter. Gel-shift analysis and chromatin immunoprecipitation assay indicated that nuclear NF-kappaB is capable of binding to this response element and that binding decreases during early stages of differentiation. Luciferase reporter gene assay demonstrated that a mutation in this binding domain leads to decreased activity. These results demonstrate that during neuronal differentiation of P19 cells, there is a decrease in specific isoforms of DMT1 via both post-translational and transcriptional mechanisms.