The alpha-MHC gene is under positive regulation by 3,5,3'-triiodo-L-thyronine (T3), however, the mechanism by which T3 modulates its transcription is not clearly understood. We have used an avidin-biotin complex DNA binding assay and footprint analysis employing dimethyl sulfate interference and hydroxyl radical protection to characterize the interaction of T3 receptors with target sequences located in the 5'-flanking region of the human alpha-myosin heavy chain (MHC) gene. The results indicate that liver T3 receptors and in vitro transcribed-translated beta c-erbA bind with high affinity to a site (TRE1) located at positions -138/-158 base pairs upstream from the CAP site. The Kd of TRE1 for nuclear liver T3 receptors (0.82 nM) was less than that determined for the rat growth hormone gene (1.78 nM). An additional site (TRE2) located at positions -111/-129 was found which bound T3 receptors with considerably lower affinity (Kd = 23 nM). Methylation interference experiments demonstrated that T3 receptors interact with guanines, in TRE1 on the sense and antisense strands, within two octameric imperfect direct repeats (underlined) 5'-TCTGGAGGTGACAG-GAGGACA-3' (antisense strand sequence) containing the consensus sequence 5'-C(T/A)GGAGG(T/A)-3'. By contrast, methylation interference and hydroxyl radical footprinting demonstrate that the T3 binding element of TRE2 is not structurally similar to TRE1 except for a purine-rich octameric cluster (underlined, 5'-ATCAAAGGAGGAGGAGCCA-3') containing six guanines on the sense strand. These results suggest that differences in nucleotide sequences involved in T3 receptor-DNA complex formation determine the binding affinities of TRE1 and TRE2.