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, TRα1 and TRβ1, 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 TRα1 and TRβ1 regulate a largely overlapping repertoire of target genes in response to T₃ hormone. However, these two isoforms display very different transcriptional properties on each individual target gene, ranging from a much greater T₃-mediated regulation by TRα1 than by TRβ1, to near equal regulation by both isoforms. We also identified TRα1 and TRβ1 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 TRα1 and TRβ1 over evolutionary time. In essence, TRα1 and TRβ1 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 T₃ response can be individually tailored to different physiological and developmental requirements.