Thyroid hormone receptors (TR) are ligand-dependent transcription factors that are encoded as multiple isoforms on two genes. To date, no functional differences have been shown between the TR isoforms; however, the maintenance of alpha and beta genes during vertebrate evolution argues that functional differences do exist. We have localized a TR-binding site in the rabbit beta-myosin heavy chain gene. This site and TR-binding sites in the chicken lysozyme and rat alpha-glycoprotein hormone genes were used to compare the DNA-binding activities of TR alpha and TR beta and the influence of auxiliary proteins (TRAP). TR alpha formed alpha/alpha homodimers poorly, whereas TR beta formed beta/beta homodimers preferentially. This difference was not due to either a lower amount of TR alpha synthesized in the reticulocyte lysate or to an inability of the expressed TR alpha to form dimers, since both TR alpha and TR beta readily formed heterodimers with TRAP on these TR-binding sites. Additionally, a TR alpha fragment containing the dimerization domains (TR alpha C291) blocks TR beta/TRAP complexes but not TR alpha/TRAP complexes. This indicates that TR beta/TR alpha dimers form more readily than TR alpha/TR alpha dimers. We conclude that on the binding sites examined, TR beta has a greater tendency to form homodimers than TR alpha, whereas both isoforms form heterodimers similarly. The different homodimerization potentials of TR alpha and TR beta may underlie functional differences that affect thyroid hormone responses.