This study is designed to clarify the role of an orphan nuclear hormone receptor, RORa, on thyroid hormone (TH) receptor (TR)- mediated transcription on a TH-response element (TRE). A transient transfection study using various TREs (i.e., F2 (chick lysozyme TRE), DR4 (direct repeat), and palindrome TRE) and TR and RORa1 was performed. When RORa1 and TR were cotransfected into CV1 cells, RORa1 enhanced the transactivation by liganded-TR on all TREs tested without an effect on basal repression by unliganded TR. By electrophoretic mobility shift assay, on the other hand, although RORa bound to all TREs tested as a monomer, no (or weak) TR and RORa1 heterodimer formation was observed on various TREs except when a putative ROR-response element was present. The transacti- vation by RORa1 on a ROR-response element, which does not contain a TRE, was not enhanced by TR. The effect of RORa1 on the TREs is unique, because, whereas other nuclear hormone receptors (such as vitamin D receptor) may competitively bind to TRE to exert dominant negative function, RORa1 augmented TR action. These results indi- cate that RORa1 may modify the effect of liganded TR on TH-re- sponsive genes. Because TR and RORa are coexpressed in cerebellar Purkinje cells, and perinatal hypothyroid animals and RORa-dis- rupted animals show similar abnormalities of this cell type, cross-talk between these two receptors may play a critical role in Purkinje cell differentiation. (Endocrinology 140: 1356 -1364, 1999)