The question of whether mature sterol regulatory element binding protein-2 (SREBP-2) mediates transcriptional activation of the hepatic low density lipoprotein (LDL) receptor by thyroid hormone was investigated. Western blotting analysis and electrophoretic mobility shift assays demonstrated that mature nuclear SREBP-2 protein could be detected in liver nuclear extracts prepared from normal animals but not in extracts prepared from rats rendered hypothyroid either by hypophysectomy (Hx) or thyroidectomy (Tx). Treatment of Hx rats with T3 restored LDL receptor mRNA levels in about 1 h and caused a 6-fold increase 2.5 h after T3 administration. However, no detectable mature SREBP-2 was seen in this time period despite a substantial reduction in serum cholesterol levels caused by the T3 treatment. Deletion of the SRE region from the LDL receptor promoter did not decrease the T3 response. Thus, the possibility that T3 may be mediating LDL receptor induction directly via a thyroid response element (TRE) was investigated. Reporter gene analysis and electrophoretic mobility shift assays demonstrated that the rat LDL receptor promoter contains two functional TREs (US-TRE and 2H-TRE). Either one of these elements could support T3 induction. However, the stronger of these elements is US-TRE at-612 which binds TRbeta1 more tightly and when mutated results in a diminished T3 response. These results indicate that the rapid induction of the hepatic LDL receptor by thyroid hormone is likely due to direct interaction with TREs rather than indirectly by a mechanism involving SREBP-2.