A cDNA encoding a novel member of the thyroid/steroid hormone receptor superfamily, called Rev-ErbAα, has been isolated from a rat GH₃ cell library. Rev-ErbAα is an ~56-kilodalton protein most similar in structure to the thyroid hormone receptor (c-erbA) and the retinoic acid receptor, but it does not bind either thyroid hormone or retinoic acid. The mRNA encoding Rev-ErbAα is present in many tissues and is particularly abundant in skeletal muscle and brown fat. A genomic DNA fragment containing the entire Rev-ErbAα cDNA sequence was isolated and characterized. Remarkably, this DNA fragment also contained a portion of the c-erbAα gene. r-erbAα-1 and r-erbAα-2 are alternative splice products of the c-erbAα gene and are members of the receptor superfamily. The genes encoding Rev-ErbAα and r-erbAα-2 overlap, with their coding strands oriented opposite one another. A 269-base-pair segment of the bidirectionally transcribed region is exonic in both the Rev-ErbAα and r-erbAα-2 genes, resulting in complementary mRNAs. Thus, through alternative splicing and opposite-strand transcription, a single genomic locus codes for three different members of the thyroid/steroid hormone receptor superfamily. Potential implications of this unusual genomic arrangement are discussed.