Thyroid hormones are major determinants of skeletal muscle differentiation In vivo. Triiodo-L-thyronlne treatment promotes terminal muscle differentiation and results In increased MyoD gene transcription in myogenic cell lines; furthermore myoD and fast myosln heavy chain gene expression are activated In rodent slow twitch muscle fibers (Molecular Endocrinology 6: 1185-1194, 1992; Development 118: 1137-1147, 1993). We have Identified a T3 response element (TRE) in the mouse MyoD promoter between nucleotide positions -337 and -309 (5' CTGAGGTCAGTACA-GGCTGGAGGAGTAGA 3'). This sequence conferred an appropriate T3 response to an enhanceriess SV40 promoter. In vitro binding studies showed that the thyroid hormone receptor α (TRα) formed a heterodimeric complex, with either the retinoid X receptor α or γ1 isoforms (RXR α, RXRγ), on the MyoD TRE that was specifically competed by other well characterised TREs and not by other response elements. Analyses of this heterodimer with a battery of steroid hormone response elements Indicated that the complex was efficiently competed by a direct repeat of the AGGTCA motif separated by 4 nucleotides as predicted by the 3-4-5 rule. EMSA experiments demonstrated that the nuclear factor(s) present In muscle cells that bound to the myoD TRE were constltutlvely expressed during myogenesis; this complex was competed by the myosin heavy chain, DR-4 and PAL-0 TREs in a sequence specific fashion. Western blot analysis Indicated that TRα1 was constitutively expressed during C2C12 differentiation. Mutagenesis of the myoD TRE indicated that the sequence of the direct repeats (AGGTCA) and the 4 nucleotide gap were necessary for efficient binding to the TRα/RXRα heterodimeric complex. In conclusion our data suggest that the TRE in the helix loop helix gene, myoD, is a target for the direct heterodimeric binding of TRα and RXRα/γ. These results provide a molecular mechanism/model for the effects of triiodo-L-thyronine on In vitro myogenesis; the activation of myoD gene expression in the slow twitch fibres and the cascade of myogenic events regulated by thyroid hormone.