This study uses fluorescence microscopy combined with dynamic video imaging to examine the events associated with the rapid desensitization of the thyrotropin-releasing hormone receptor (TRH-R). In single non-pituitary human embryonic kidney 293 (HEK-293) cells, expressing either the rat or human TRH-Rs, TRH produced a rapid dose-dependent monophasic rise in [Ca2+]i. This Ca2+ transient was completely abolished by pretreatment of cells with the intracellular Ca2+ antagonists thapsigargin or cyclopiazonic acid, but not EGTA, the voltage-operated Ca2+ channel (VOCC) antagonist nifedipine or the second-messenger-operated Ca2+ channel antagonist SK&F 96365. These results suggest that TRH causes the mobilization of Ca2+ from thapsigargin/cyclopiazonic acid-sensitive intracellular Ca2+ stores but not the influx of extracellular Ca2+. HEK-293 cells also failed to respond to KCl or the slow Ca(2+)-channel activator BAY K 8644, suggesting that they lack L-type VOCCs. Rat and human TRH-Rs are highly conserved except at the C-terminus where the sequence differs. The C-terminus is believed to be important in receptor desensitization. Despite differences in this region, rat and human TRH-Rs expressed in HEK-293 cells underwent rapid (within 1 min) desensitization. This desensitization was dose-dependent and did not involve receptor loss. Similarly the bradykinin receptor endogenous to HEK-293 cells also displays a rapid desensitization. We conclude that in TRH-R-expressing non-pituitary HEK-293 cells, TRH mobilizes intracellular Ca2+ resulting in a monophasic Ca2+ transient. The rat and human TRH-Rs as well as the endogenous bradykinin receptor also displayed rapid receptor desensitization.