DNA binding and mutagenesis in vitro established that the −67/−55 region of the apoA-II (apolipoprotein A-II) promoter contains a thyroid HRE (hormone response element), which strongly binds RXRα (retinoid X receptor α)/T3Rβ (thyroid receptor β) heterodimers and weakly T3Rβ homodimers, but does not bind other homo- or heterodimers of RXRα or orphan nuclear receptors. Transactivation was abolished by point mutations in the thyroid HRE. In co-transfection experiments of HEK-293 (human embryonic kidney 293) cells, the −911/+29 human apoA-II promoter was transactivated strongly by RXRα/T3Rβ heterodimers in the presence of RA (9-cis retinoic acid) or T3 (tri-iodothyronine). Homopolymeric promoters containing either three copies of the −73/−40 (element AIIAB) or four copies of the −738/−712 (element AIIJ) apoA-II promoter could be transactivated by RXRα/T3Rβ heterodimers in COS-7 cells only in the presence of T3 or RA respectively. RXRα/T3Rβ heterodimers and USF2a (upstream stimulatory factor 2a) synergistically transactivated the −911/+29 apoA-II promoter in the presence of T3. USF2a also enhanced the activity of a GAL4–T3Rβ fusion protein in the presence of T3 and suppressed the activity of a GAL4–RXRα fusion protein in the presence of RA. These findings suggest a functional specificity of the two HREs of the apoA-II promoter for retinoids and thyroids, which is modulated by synergistic or antagonistic interactions between RXRα/T3Rβ heterodimers and the ubiquitous transcription factor USF2a.