These studies were designed to characterize ubiquitination of the G protein-coupled TRH (TRH) receptor. TRH receptors and ubiquitin co-precipitated with antibodies to either receptor or ubiquitin in CHO or pituitary GHFT cells. Inhibition of the proteasome with MG-132 resulted in an accumulation of total TRH receptors and the appearance of a small amount of cytosolic receptor. MG-132 caused an increase in newly synthesized receptors, detected by microscopy using a TRH receptor coupled to Timer, a DsRed that undergoes a spontaneous time-dependent color change. Misfolded TRH receptors were particularly heavily ubiquitinated. These results show that the proteasome participates in TRH receptor quality control early after receptor synthesis. Under normal circumstances, most ubiquitinated TRH receptors were absorbed to wheat germ agglutinin, indicating that they had undergone complex glycosylation in the Golgi apparatus. When cells were treated with tunicamycin to block glycosylation, a ladder of ubiquitinated species was detectable. Cell surface receptors, which were labeled selectively with either radioligand or antibody, showed no detectable ubiquitin modification. To determine if ubiqutination plays a role in TRH-induced receptor endocytosis, the receptor was expressed in Ts20 cells, which have a temperature-sensitive ubiquitin pathway. TRH induced a significant calcium response and rapid and extensive receptor internalization at both the permissive and non-permissive temperatures, indicating that ligand dependent ubiquitination of the receptor, or any other protein, is not necessary for TRH receptor signaling or internalization. These results show that ubiquitin modification targets misfolded receptors for degradation and suggest a possible role for ubiquitination in receptor trafficking.