We examined the molecular basis by which T3 reg- ulates the human cholesterol 7-hydroxylase gene (CYP7A1) promoter. L-T3 decreased chloramphen- icol acetyltransferase activity in hepatoma cells cotransfected with a plasmid encoding the T3 re- ceptor (TR) (NR1a1) and a chimeric gene con- taining nucleotides 372 to 61 of the human CYP7A1 gene fused to the chloramphenicol acetyl- transferase structural gene. Deoxyribonuclease I footprinting revealed that recombinant TR pro- tected two regions in this segment of the human CYP7A1 gene promoter. In EMSAs, TR bound to both regions. The binding was competed by oligo- nucleotides bearing an idealized TR binding motif and abolished by mutation of these elements. In assays of promoter function, mutation of only one of the TR binding sites blocked repression by T3. The results indicate that T3-dependent repression of human CYP7A1 gene expression is mediated via a novel site in the human CYP7A1 gene promoter. (Molecular Endocrinology 16: 14-23, 2002)