We have used thyroid hormone receptors from two different human cell lines to investigate receptor binding to the promoters of thyroid hormone-responsive genes. Receptors extracted from IM-9 cells or HeLa cells displayed virtually identical affinity and specificity for [125I]triiodothyronine binding. The cells expressed a c-erbA alpha gene in the same relative proportions as the receptor concentrations. Both receptors were bound to DNA-cellulose and could be displaced with increasing concentrations of calf thymus DNA or pBR322 DNA. Relative to pBR322 DNA (designated as 1), binding to the hGH gene promoter was 8.1 +/- 1.1 using the IM-9 cell receptor. With the HeLa cell receptor relative binding was only 1.1 +/- 0.2. Similar relative differences were obtained with the mouse glandular kallikrein gene, mGK-6. In heat stability studies the IM-9 cell receptor was more resistant to heat inactivation than the HeLa receptor. Triiodothyronine receptors with identical hormone binding patterns may require the presence of an unidentified factor(s) which allows correct recognition of regulation sequences within responsive genes.