A putative Ets site with a core of GGAA located at -88 to -85 of the rat ornithine decarboxylase (ODC) gene was characterized by site-directed mutagenesis and transient expression assays. Mutation of this site, when in pODClux2m which contains a cluster of four Sp1 binding sites, resulted in a 2.6-fold increase in basal promoter activity in untreated cells, while the ratio of activity in 12-O-tetradecanoylphorbol-13-acetate (TPA) treated cells relative to that in untreated cells remained (the induction ratio) was largely unchanged. However, when the mutation is in pODClux168, which contains only a single Sp1 binding site (GC box V), it caused little alteration to either basal promoter activity or TPA induction ratio. A protein of 55-60kDa was found specifically bound to this site, as revealed by UV-cross linking assay. In competition assay and methylation interference assay, this protein was shown to occupy the GGAA core, although it showed no antigenic relation to c-Ets-1 in supershift assay. We suggest that this protein binds specifically to the GGAA core, and functions to inhibit activation of the ODC promoter by distal elements, including the upstream Sp1 sites.