Neural zinc finger factor 1 (NZF-1), a transcription factor important for the development of the nervous system, is a member of a protein family characterized by the presence of one or more copies of a CCHHC type zinc binding domain. These domains fold around a single zinc ion to generate a compact, looped structure. A minimum of two zinc binding domains are required for appreciable DNA binding to be observed. While zinc is the presumptive metal utilized in vivo, several studies of other classes of zinc-binding domain have shown that other metals are capable replacing zinc in vitro. Disruption of iron homeostasis has been implicated in a number of neurodegenerative diseases, including Alzheimer, Huntington and Parkinson diseases. Given its important role in the developing nervous system, we investigated the ability of iron to substitute for zinc within the zinc-binding domains of NZF-1, to assess the affect of iron substitution on DNA binding. Using UV-vis spectroscopy, we determined dissociation constants (K_d) for zinc(II) and iron(III) binding to a fragment of NZF-1 containing two zinc-binding domains, and found them to be 1.3x10^-10M and 2.3x10^-5M, respectively. The DNA binding affinity of this protein fragment was examined through fluorescence anisotropy, using a fluorescently labeled 16bp oligonucleotide representing the b-retinoic acid receptor element (b-RARE), a known DNA binding site for NZF-1. The iron(II)- and iron(III)-NZF-1 complexes bound DNA with dissociation constants of 2.5x10^-5M and 3.1x10^-5M, while the zinc-bound form of the protein had a K_d of 4.7x10^-5M. We conclude that while the zinc-binding domains bind iron with 10⁵ fold weaker affinity, the substitution of iron for zinc does not disrupt the DNA binding ability of the protein. Future investigations will examine if localization of the redox-active iron to DNA in this manner leads to DNA damage via Fenton chemistry.