Thyroid hormone receptors (TRs) are hormone-regulated transcription factors that control multiple aspects of physiology and development. TRs are expressed in vertebrates as a series of distinct isoforms that exert distinct biological roles. We wished to determine whether the two most widely expressed isoforms, TR alpha 1 and TR beta 1, exert their different biological effects by regulating different sets of target genes. Using stably transformed HepG2 cells and a microarray analysis, we were able to demonstrate that TR alpha 1 and TR beta 1 regulate a largely overlapping repertoire of target genes in response to T(3) hormone. However, these two isoforms display very different transcriptional properties on each individual target gene, ranging from a much greater T(3)-mediated regulation by TR alpha 1 than by TR beta 1, to near equal regulation by both isoforms. We also identified TR alpha 1 and TR beta 1 target genes that were regulated by these receptors in a hormone-independent fashion. We suggest that it is this gene-specific, isoform-specific amplitude of transcriptional regulation that is the likely basis for the appearance and maintenance of TR alpha 1 and TR beta 1 over evolutionary time. In essence, TR alpha 1 and TR beta 1 adjust the magnitude of the transcriptional response at different target genes to different levels; by altering the ratio of these isoforms in different tissues or at different developmental times, the intensity of T(3) response can be individually tailored to different physiological and developmental requirements.