We developed a sensitive competitive RT-PCR technique for quantitating the ratio of D2 to cyclophilin messenger RNA (mRNA) and used this to study type 2 deiodinase (D2) mRNA regulation. Hyperthyroidism in rats causes a 2- to 3-fold reduction in anterior pituitary and medial basal hypothalamus (MBH). Thyroid hormone (T₃) withdrawal increased the D2/cyclophilin ratio 2- to 3-fold over 48 h in both GC and GH4C1 cells. T₃ additional reduced D2 gene transcription by 50% over 2 h and about 30% over the next 2 h. D2 mRNA half-life is 2 h and is not affected by T₃, indicating that its effect is due to suppression of D2 gene transcription. The T₃ effect did not require new protein synthesis. Longer treatment with T₃ led to a maximum decrease of 70% in D2 mRNA, indicating that there is also a T₃-independent transcriptional component of the D2 gene. 3,3′,5′-Triiodothyronine (reverse T₃) caused a slight increase D2 mRNA over 24 h but an 80–90% decrease in D2 activity, indicating that it acts posttranscriptionally. Dexamethasone, 8 Br-cAMP, and TRH also caused modest increases in D2 mRNA in pituitary tumor cells. We conclude that D2 gene transcription has both T₃-dependent and T₃-independent components. Thus, posttranscriptional effects of D2 substrates such as T₄ will be required for complete feedback inhibition of D2 activity. The short half-life of D2 mRNA and D2 protein explains the rapid response of D2 activity to thyroid hormone administration.