Ear3/COUP-TF belongs to the steroid/thyroid hormone receptor superfamily of nuclear transcription factors. Previous studies have identified six target response elements to which the factors of the family bind with various affinities. They are palindromic pair of hexameric half site (Thyroid hormone Response Element; TRE, Estrogen Response Element; ERE, Glucocorticoid Response Element; GRE) or directly repeated half sites [Direct Repeat (DR) 3, DR4, DR5]. In this report, these six elements and three additional putative elements, DR0, DR1, and DR2, which are directly repeated half sites separated by 0, 1 and 2 nucleotides, respectively, were tested for binding to Ear3/COUP-TF protein employing gel retardation method. Among the palindromic series of response elements, TRE showed the highest affinity, ERE showed second highest, and no binding was observed for GRE. Among six direct repeat response elements, DR1 showed the highest affinity. Comparison of binding affinities between DR1 and TRE revealed that the DR1 has the highest affinity of all the nine response elements tested. The consensus sequence of the DR1 element resembles well the COUP sequence found in the upstream promoter of chicken ovalbumin gene, which has been shown to serve as a natural response element for Ear3/COUP-TF. Retinoid X receptor (RXR), another member of the receptor family, reportedly utilizes DR1 sequence as its response element, suggesting that Ear3/COUP-TF could regulate the expression of the same subsets of genes as RXR does.