The conserved diversity, restricted distribution, and differential regulation of the thyroid hormone receptor (TR) isoforms raise the possibility of isoform-specific functions. We have addressed the roles of individual TRs in GH gene expression in GH3 cells by using an isoform-specific antisense RNA to delete TRβ1. An antisense RNA vector, directed against the isoform-specific coding sequence of the parent TRβ1 complementary DNA, was constructed. Stable transfected GH₃-derived cell lines expressing this construct were established. Appropriate control cell lines were established in parallel. Depletion of TRβ1 in cells expressing the antisense construct was confirmed at both the messenger RNA and protein levels. Total TR expression was maintained in these cells by a reciprocal increase in TRβ2 levels. This perturbation of the TR population was associated with a 10.5-fold increase in basal and a 5.0-fold increase in T₃-stimulated GH gene expression, but no increase in total T₃ binding of nuclear extracts. In transient cotransfection experiments, there were no differences between control cells and those expressing the antisense construct in either basal or T₃-stimulated expression of reporters containing a variety of thyroid hormone response elements. Depletion of TRβ1 in GH₃ cells results in a reciprocal increase in TRβ2. These changes are associated with increased basal and T₃-stimulated GH gene expression, which are not due to a nonspecific enhancement of basal or hormone-stimulated transcription. We demonstrate that TRβ1 is not required for T₃ induction of the GH gene in GH₃ cells and that TRβ1 and TRβ2 are not equivalent in their effects on basal repression of the GH promoter. The data illustrate the potential for isoform- and promoter-specific dissociation of the repression and activation properties of the TRs.