To better delineate the sequence requirements for high affinity binding of retinoid X receptor α (RXRα) homodimers, a selection protocol was used starting from a random pool of oligonucleotides. All recovered sequences contained at least two hexamers related to the consensus sequence for the thyroid/retinoid subfamily of nuclear receptors, A GGGTCA . These hexamers were most frequently organised as direct repeats with one interspacing base pair (DR1) and as palindromic repeats without interspacing base pairs (PAL0), the established configurations for RXR response elements (RXREs). However, DR2 and DR6 configurations also appeared to bind RXRα homodimers with high affinity, as did elements consisting of three hexamers. Reporters containing single copies of these elements conferred 9-cis retinoic acid responsiveness to cells cotransfected with an RXRα expressing plasmid. The upstream hexamer of all recovered sites was preferentially preceded by a G and its consensus was GGGTCA. Based on the composition of the selected DR1 RXREs, and the functional and mutational analysis, the optimal DR1 RXRE consists of an upstream hexamer starting with A or G and preceded by A or G. The interspacing base can be either G, A or T but not C. The affinity of RXRα homodimers for a DR1 element is strongly reduced when the final position is taken by a C. The results of the present investigation indicate that RXRα homodimers may have broader DNA binding specificities than currently believed. The biological relevance of these alternative RXREs will need to be corroborated by the identification of natural elements of this kind.