The mouse mammary tumor virus promoter has been shown to be inducible by glucocorticoids and progesterone. Although steroid hormone receptors bind with high affinity to palindromic response elements, the hormone-responsive region of the mouse mammary tumor virus promoter contains a pair of directly repeated half-sites that are important for hormone inducibility. Recent experiments have also indicated that direct repeats can function as estrogen response elements. Here, we have investigated DNA binding by steroid receptors to direct repeats and provide evidence using gel retardation assays, methylation interference, and gene transfer experiments that direct repeats of TGTTCT or RGGTCA motifs function as response elements for glucocorticoid (GR) or estrogen receptors (ER), respectively, by binding receptor homodimers. Specific GR- or ER-DNA complexes were observed on direct repeats with different spacings between half-sites, indicating that binding of steroid receptors to direct repeats is more flexible than binding to palindromic elements. This flexibility was further emphasized by the observation that the GR could also bind to everted repeats of TGTTCT motifs separated by 9 base pairs. The isolated DNA binding domains of the GR and ER bound cooperatively to palindromes, but no evidence was observed for cooperative binding to direct repeats. Under similar conditions the DNA binding domains of retinoid receptors retinoid X receptor and retinoic acid receptor bound to direct repeats cooperatively as heterodimers. Similarly, ER derivative HE15, which lacks a functional ligand binding domain, bound palindromic response elements but failed to bind direct repeats. These results indicate that the dimerization domain in the ligand binding domain is essential for binding of steroid receptors to direct repeats and that the dimerization domain in the D-box of the DNA binding is not functional under these conditions. Moreover, the results suggest that steroid receptor DNA binding domains may lack dimerization domains outside the D-box, which would function in binding to direct repeats, in contrast to receptors for retinoids and thyroid hormone. A comparison of the mechanisms of binding of steroid receptors and retinoid and thyroid hormone receptors to direct repeats is presented.