Binding of semi-purified rat liver triiodothyronine (T3) nuclear receptor (T3-nR) to sequences in the 5' flanking DNA of rat growth hormone (rGH) has been evaluated by the DNAse-footprinting technique. T3-nR was obtained by extraction in 0.3 M KCl and partially purified by column chromatography to 80 pmol/mg T3 maximal binding capacity (MBC). The T3-nR preparation protects the sequence -330 to -314 (5'-CAGTGAACAAACGATG-3') which may represent a repressor element, the sequence -224 to -192 (5'-CATTGCCCATAAACCTGAGC-3') which is thought to involve positive T3 regulation, the sequence -98 to -78 (5'-GCTCCAGCCATGAATAAATG-3') which may regulate basal expression, and the TATA box area. Although the DNAse footprints occur only with preparations containing functional T3-nR, it is uncertain whether each protected zone is due to binding of T3-nR or associated proteins. Protection is associated with enhanced DNAse digestion around the protected sites, involves both strands, and does not appear to require T3 occupancy of the receptor. Only the areas described are protected in the studies, and non-specific proteins such as albumin, core histones, and nuclear extracts without receptor activity, have no effect. A putative consensus enhancer sequence -CCAGAGCCAAGG- conveying T3 responsivity is suggested on the basis of comparison to sequences in four T3 responsive genes.