Appropriate cellular levels of polyamines are required for cell growth and differentiation. Ornithine decarboxylase is a key regulatory enzyme in the biosynthesis of polyamines, and precise regulation of the expression of this enzyme is required, according to cellular growth state. A variety of mitogens increase the level of ornithine decarboxylase activity, and, in most cases, this elevation is due to increased levels of mRNA. A GC box in the proximal promoter of the ornithine decarboxylase gene is required for basal and induced transcriptional activity, and two proteins, Sp1 and NF-ODC1, bind to this region in a mutually exclusive manner. Using a yeast one-hybrid screening method, ZBP-89, a DNA-binding protein, was identified as a candidate for the protein responsible for NF-ODC1 binding activity. Three lines of evidence verified this identification; ZBP-89 copurified with NF-ODC1 binding activity, ZBP-89 antibodies specifically abolished NF-ODC1 binding to the GC box, and binding affinities of 12 different double-stranded oligonucleotides were indistinguishable between NF-ODC1, in nuclear extract, and in vitro translated ZBP-89. ZBP-89 inhibited the activation of the ornithine decarboxylase promoter by Sp1 in Schneider's Drosophila line 2, consistent with properties previously attributed to NF-ODC1.