In addition to its well known actions in stimulating TSH and PRL synthesis and secretion, TRH has been shown to decrease the concentration of thyroid hormone receptors (TRs) in GH4Ci cells as measured by nuclear thyroid hormone (T₃) binding. In the present study we have investigated the effects of TRH on the levels of mRNA encoding the different forms of TR, TRβ-1, TRβ-2, and TRa-1 as well as that of the non-T₃-binding variant, c-erbAa-2. GH3 cells were incubated with 100 nM TRH in the presence or absence of 1 nM T₃ for 48 h, and mRNA levels were determined by Northern blot analysis. Results revealed that there is differential regulation of the individual TRs by TRH at the pretranslational level. The mRNA for the pituitary-specific form of TR, TRβ-2, was down-regulated by 60% by TRH in GH3 cells, while that of its alternative splice product, TRβ-1, was unchanged. A modest change was observed in TRa-1 mRNA levels, which were down-regulated by 20%; there was no change in c-er…Aa-2 mRNA levels. Levels of nuclear T₃ binding were assessed under the same conditions, and 100 nM TRH was found to decrease binding by 40% from 0.78 to 0.46 fmol/jig DNA. A similar change in nuclear T₃ binding was seen after incubation with 1 nM T₃. The effect of TRH on the GH mRNA response to T₃ was investigated. In the absence of TRH there was a 4-fold induction of GH mRNA after incubation with 1 nM T₃. In the presence of 100 nM TRH, no significant induction in GH mRNA by T₃ was seen, indicating that T₃ responsiveness as well as receptor concentration are diminished by TRH under these conditions. (Endocrinology128: 1763—1768, 1991)