The cytoplasmic iron regulatory protein (IRP) modulates iron homeostasis by binding to iron-responsive elements (IREs) in the transferrin receptor and ferritin mRNAs to coordinately regulate transferrin receptor mRNA stability and ferritin mRNA translational efficiency, respectively. These studies demonstrate that thyroid hormone (T-3) can modulate the binding activity of the IRP to an IRE in vitro and in vivo. T-3 augmented an iron-induced reduction in IRP binding activity to a ferritin IRE in RNA electrophoretic mobility shift assays using cytoplasmic extracts from human liver hepatoma (HepG2) cells. Hepatic IRP binding to the ferritin IRE also diminished after in vivo administration of T-3 with iron to rats. In transient transfection studies using HepG2 cells and a human ferritin IRE-chloramphenicol acetyltransferase (H-IRE-CAT) construct, T-3 augmented an iron-induced increase in CAT activity by similar to 45%. RNase protection analysis showed that this increase in CAT activity was not due to a change in the steady state level of CAT mRNA. Nuclear T-3-receptors may be necessary for this T-3-induced response, because the effect could not be reproduced by the addition of T-3 directly to cytoplasmic extracts and was absent in CV-1 cells which lack T-3-receptors. We conclude that T-3 can functionally regulate the IRE binding activity of the IRP. These observations provide evidence of a novel mechanism for T-3 to up-regulate hepatic ferritin expression, which may in part contribute to the elevated serum ferritin levels seen in hyperthyroidism.