c-erbA cDNAs encoding thyroid hormone-binding receptor proteins have been previously isolated from several species and tissues. We have isolated a novel rat c-erbA cDNA, r-erbAα-2, identical to the rat brain c-erbAα cDNA (which we refer to as r-erbAα-1) except at the 3′-end of the cDNA, which encodes an altered carboxy-terminal region of the predicted amino acid sequence. The r-erbAα-2 cDNA is most closely related to the human testicular c-erbAα cDNA. The apparent molecular weight of the in vitro protein product of this cDNA is 55 K, close to that predicted from the nucleotide sequence. The r-erbAα-2 protein binds a DNA fragment containing a putative T₃-responsive sequence from the rat GH gene. However, the r-erbAα-2 protein product does not bind T₃. RNA isolated from rat GH₃ cells and various rat tissues contains at least three mRNAs hybridizing to c-erbAα probes. A predominant 2.6 kilobase mRNA hybridizes specifically to r-erbAα-2, while a 5.0 kb mRNA hybridizes to r-erbAα-1-specific cDNA probes. The r-erbAα-1-related 5.0 kb mRNA is down-regulated by T₃ treatment in GH₃ cells as has previously been described for the 2.6 kb mRNA. The r-erbAα-2 mRNA is most abundant in brain, whereas the r-erbAα-1 mRNA is found in highest concentration in brown fat and skeletal muscle. r-erbAα-1 mRNA, unlike r-erbAα-2 mRNA, is undetectable in testis. Southern analyses suggest that the r-erbAα-1 and r-erbAα-2 mRNAs are derived from a single gene transcript.