Gel shift assays have been employed to examine the association of the thyroid hormone receptor with specific DNA sequences in the 5'-flanking DNA of the rat growth hormone (rGH) gene. This DNA is known to have structure(s) that mediate thyroid hormone effects on the rGH promoter. The receptors used were obtained from preparations purified 300-500-fold from rat liver nuclear extracts and contained about 1% pure receptors. Thyroid hormone receptor binding to DNA was assessed by monitoring protein-bound 32P-labeled restriction endonuclease fragments in parallel with L-tri[125I]iodothyronine-labeled protein-DNA complexes. The receptors were found to bind specifically to four different regions of the rGH 5'-flanking DNA (nucleotides -1730 to -1230, -530 to -230, -181 to -149, and -149 to +12) numbered with respect to the transcriptional start site. The specificity of the binding was documented by the finding that the receptor did not bind to other rGH 5'-flanking DNA sequences or to several other DNAs and by the fact that only the DNAs exhibiting specific binding could block the binding of radiolabeled DNA. The binding was also detected in NaCl concentrations up to 140 mM, reduced by Mg2+ concentrations up to 5 mM, and inhibited by 1 mM zinc. The DNA sequence-specific binding of the receptor was found to require occupancy of the receptor by the hormone (L-triiodothyronine) and could also be observed when the receptor was occupied by the thyroid hormone antagonist amiodarone. These results indicate that thyroid hormone receptors interact specifically with several sites on the 5'-flanking DNA of the rGH gene and that hormone occupancy is not required for the binding. Thus, thyroid hormone may act by stimulating a transcriptional activation function of the receptor rather than by stimulating DNA binding per se.