In all vertebrates, thyroid hormones (TH) affect postembryonic development. The role of TH receptor (TR) in mediating TH signal is complex as evidenced by divergent phenotypes in mice lacking TH compared to TR knockout mice. We have proposed a dual function model for TR during development based on studies of frog metamorphosis. Here, we examined an important assumption of this dual function model using the chromatin immunoprecipitation (ChIP) assay, namely constitutive TR binding to promoters in vivo. We examined two target genes with TH response elements (TRE) in their promoters, TRß itself and TH/bZIP (TH-responsive basic leucine zipper transcription factor). Using an antibody that recognizes both TRa and TRß, we found that TR binding to TRß promoter is indeed constitutive. Surprisingly, TR binding to TH/bZIP promoter increases dramatically after TH treatment of premetamorphic tadpoles and during metamorphosis. Using an antibody specific to TRß, TRß binding increases at both promoters in response to TH. In-vitro biochemical studies showed that TRs bind TH/bZIP TRE with 4-fold lower affinity than to TRß TRE. Our data, showing that only high affinity TRß TRE is occupied by limiting levels of TR during premetamorphosis and that lower affinity TH/bZIP TRE becomes occupied only when overall TR expression is higher during metamorphosis, provide the first in vivo evidence to suggest that one mechanism for tissue and gene-specific regulation of TR target gene expression is through tissue and developmental stage-dependent regulation of TR levels, likely a critical mechanism for coordinating development in different organs during postembryonic development.