A carboxyl-terminus truncated mutant of the guanine nucleotide-binding (G) protein-coupled TRH receptor (TRH-R) was previously shown to exhibit constitutive, i.e. TRH-independent, activity (C335Stop TRH-R). Chlordiazepoxide (CDE), a known competitive inhibitor of TRH binding to wild-type (WT) TRH-Rs, is shown to compete for binding to C335Stop TRH-Rs also. More importantly, CDE is shown to be a negative antagonist of C335Stop TRH-Rs. CDE rapidly caused the basal rate of inositol phosphate second messenger (IP) formation to decrease in AtT-20 pituitary cells stably expressing C335Stop TRH-Rs (AtT-C335Stop cells), but not in cells expressing WT TRH-Rs (AtT-WT cells). Similar observations were made in HeLa cells transiently expressing C335Stop or WT TRH-Rs. CDE inhibition of IP formation was shown to be specific for TRH-Rs using GH4C1 cells expressing both TRH-Rs and receptors for bombesin. In these cells, CDE inhibited TRH-stimulated IP formation, but had no effect on bombesin-stimulated IP formation. The effects of chronic administration of CDE were studied. Preincubation of AtT-C335Stop cells, but not AtT-WT cells, with CDE for several hours caused an increase in cell surface receptor number (up-regulation) that led to increased TRH stimulation of inositol phosphate formation and elevation of intracellular free Ca2+. Preincubation with CDE did not affect methyl-TRH binding affinity or TRH potency in cells expressing AtT-C335Stop or in AtT-WT cells. We conclude that CDE is a negative antagonist of C335Stop TRH-Rs and that constitutively active C335Stop TRH-Rs are down-regulated in AtT-20 pituitary cells in the absence of agonist.