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Rev-ErbA alpha (Rev-Erb) is a nuclear hormone receptor-related protein encoded on the opposite strand of the alpha-thyroid hormone receptor (TR) gene. This unusual genomic arrangement may have a regulatory role, but the conservation of human and rodent Rev-Erb amino acid sequences suggests that the protein itself has an important function, potentially as a sequence-specific transcriptional regulator. However, despite its relationship to the TR, Rev-Erb bound poorly to TR binding sites. To determine its DNA-binding specificity in an unbiased manner, Rev-Erb was synthesized in Escherichia coli, purified, and used to select specific binding-sites from libraries of random double-stranded DNA sequences. We found that Rev-Erb binds to a unique site consisting of a specific 5-bp A/T-rich sequence adjacent to a TR half-site. Rev-Erb contacts this entire asymmetric 11-bp sequence, which is the longest nonrepetitive element specifically recognized by a member of the thyroid/steroid hormone receptor superfamily, and mutations in either the A/T-rich or TR half-site regions abolished specific binding. The binding specificity of wild-type Rev-Erb was nearly identical to that of C- and N-terminally truncated forms. This binding was not enhanced by retinoid X receptor, TR, or other nuclear proteins, none of which formed heterodimers with Rev-Erb. Rev-Erb also appeared to bind to the selected site as a monomer. Furthermore, Rev-Erb activates transcription through this binding site even in the absence of exogenous ligand. Thus, Rev-Erb is a transcriptional activator whose properties differ dramatically from those of classical nuclear hormone receptors, including the TR encoded on the opposite strand of the same genomic locus.