The mammalian thyroid hormone receptor gene c-erbAα gives rise to two mRNAs that code for distinct isoforms, TRα1 and TRα2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRα1-specific polyadenylation site or TRα2-specific 5′ splice site. A previous investigation of TRα minigene expression defined a critical role for the TRα2 5′ splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEα2, enhance splicing of TRα2 in vitro as well as in vivo. Although SEα2 is located within the intron of TRα2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEα2 functions by binding trans-acting factors in HeLa nuclear extract. Protein–RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEα2. SEα2 also includes an element resembling a 5′ splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5′ splice site sequence have a dramatic effect on splicing and protein binding. Thus SEα2 and its associated factors are required for splicing of TRα2 pre-mRNA.