Transcription of the antiatherogenic protein apolipoprotein AI is regulated by the thyroid hormone, L-triiodothyronine. Transient transfection and electrophoretic mobility shift assays were used to identify the cis-acting elements involved. In transient transfection assays, hormone bound to either thyroid hormone receptor alpha or beta exerts a positive effect through a thyroid hormone response element, site A (-208 to -193). In the absence of site A, liganded receptor alpha or beta have a negative effect on promoter activity. This negative effect is mediated by a 40 bp fragment spanning nucleotides -46 to -7. Closer examination of this region of the gene shows there to be a negative thyroid hormone response element at position -25 to -20 which is fused to the 3' end of the TATA element. Electrophoretic mobility shift assays show that bacterially expressed chicken or rat thyroid hormone receptor alpha 1 binds to site A, either as a homodimer or as a heterodimer with the human 9-cis-retinoic acid receptor alpha. In contrast, the negative thyroid hormone responsive element binds chicken thyroid hormone receptor alpha exclusively as a monomer. Site-directed mutagenesis of the negative thyroid hormone response element abolished the inhibitory effects of the hormone and increased basal promoter activity by up to 40-fold. These data suggest that functional positive and negative thyroid hormone response elements coexist within the rat apolipoprotein AI promoter and both elements contribute to the control of apolipoprotein AI gene expression.