High-cholesterol diets elicit changes in gene expression via such transcription factors as sterol-regulatory element binding proteins (SREBPs) and liver X receptors (LXRs). We used Affymetrix microarrays to identify genes in mouse liver regulated by dietary cholesterol (0.0% vs. 0.5% cholesterol wt/wt). Three independent experiments were performed, and data were analyzed with Affymetrix Microarray Suite and ANOVA statistical software. There were 69 unique Unigene clusters consistently regulated by dietary cholesterol (37 downregulated and 32 upregulated). The array results were confirmed by quantitative RT-PCR (Q-PCR) for seven of nine downregulated genes and five of six upregulated genes. A time course of dietary cholesterol feeding over 1 week revealed different temporal patterns of gene regulation for these confirmed genes. Six downregulated genes were examined in transgenic mice overexpressing truncated nuclear forms of SREBP-1a and SREBP-2, and all were induced in these mice. A second microarray analysis of mice treated with the LXR agonist TO901317 confirmed that 13 of the 32 cholesterol upregulated genes were also LXR-activated. This array result was confirmed by Q-PCR for three of three genes. In summary, these studies identified and confirmed six novel dietary cholesterolregulated genes, three putative SREBP target genes (calcium/calmodulin-dependent protein kinase 1D, fatty acid binding protein 5, and proprotein convertase subtilisin/ kexin 9), and three putative LXR target genes (a disintegrin and metalloprotease domain 11, apoptosis-inhibitory 6, and F-box-only protein 3). Cholesterol is an important constituent of cellular membranes and serves as a precursor in the formation of bile acids and steroid hormones. Excessive cholesterol, however, is involved in atherosclerotic lesion and gallstone formation. Therefore, a balance must be maintained between cholesterol absorption and excretion and endogenous cholesterol synthesis. In this regard, the liver plays an important role. For example, increasing dietary cholesterol results in downregulation of lipoprotein receptors such as the LDL receptor and cholesterol biosynthetic enzymes like HMG-CoA reductase (HMGCR) and results in upregulation of the bile acid biosynthetic enzyme cholesterol 7-␣ -hydroxylase (CYP7A1) (1, 2).Cholesterol can regulate expression at both transcriptional and posttranscriptional levels, and in the former, two key transcription factors have been implicated, sterolregulatory element binding protein (SREBP) and the liver X receptor (LXR) (3, 4). The SREBP pathway utilizes three transcription factors, SREBP-1a, -1c, and -2, which are synthesized as transmembrane precursors in the endoplasmic reticulum with an N-terminal basic helix-loop-helix-Zip transcription factor domain facing the cytoplasm. When cellular sterols are low, the SREBP cleavage-activating protein (SCAP)-INSIG-1 complex brings the SREBP to the Golgi, where the site 1 protease cleaves intraluminally and the site 2 protease cleaves intramembraneously to release...