Incubation with TRH causes a decrease (over 2-24 h) in the number of TRH receptors (TRHRs) in GH4C1 rat pituitary cells. Using a homologous cDNA probe prepared from the TRHR cloned from these cells, we demonstrate here that TRH modulates the rate of transcription of TRHR mRNA. Incubation of GH4C1 cells for 4 h with TRH caused an 80% reduction in TRHR mRNA concentration. The TRHR mRNA level remained low for 48 h in the continued presence of TRH. Run-on transcription assays demonstrated that TRH caused a decrease in TRHR mRNA transcription of about 60% at 4 h. The rate of transcription remained low at 12 h. Inhibition of protein synthesis did not prevent the TRH-induced decrease in TRHR mRNA. Activation of protein kinase C with the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate decreased TRHR mRNA by about 40% in 4 h. Elevation of [Ca2+]i with ionomycin decreased TRHR mRNA by about 25%. The two agents together decreased TRHR mRNA by approximately 70% in 4 h, an effect comparable to that elicited by TRH. We conclude that regulation of endogenous TRHR mRNA by TRH is modulated, at least in part, at the level of transcription by a mechanism that probably involves both activation of protein kinase C and an increase in [Ca2+]i.