The liver is an important target organ of thyroid hormone. However, only a limited number of hepatic target genes have been identified, and little is known about the pattern of their regulation by thyroid hormone. We used a quantitative fluorescent cDNA microarray to identify novel hepatic genes regulated by thyroid hormone. Fluorescent-labeled cDNA prepared from hepatic RNA of T3-treated and hypothyroid mice was hybridized to a cDNA microarray, representing 2225 different mouse genes, followed by computer analysis to compare relative changes in gene expression. Fifty five genes, 45 not previously known to be thyroid hormone-responsive genes, were found to be regulated by thyroid hormone. Among them, 14 were positively regulated by thyroid hormone, and unexpectedly, 41 were negatively regulated. The expression of 8 of these genes was confirmed by Northern blot analyses. Thyroid hormone affected gene expression for a diverse range of cellular pathways and functions, including gluconeogenesis, lipogenesis, insulin signaling, adenylate cyclase signaling, cell proliferation, and apoptosis. This is the first application of the microarray technique to study hormonal regulation of gene expression in vivo and should prove to be a powerful tool for future studies of hormone and drug action.