Quality control in the secretory pathway limits forward transport of newly synthesized cargo proteins to those that have acquired their fully folded conformation. To determine which organelles participate in this conformation-dependent sorting process, we analyzed the trafficking of the temperature-sensitive, thermo-reversible folding mutant of vesicular stomatitis virus glycoprotein (tsO45 G protein) in VERO cells. Using temperature blocks, the G protein could be localized to the ER (39.5 degrees C), to the vesiculo-tubular clusters (VTCs, 15 degrees C), and to the trans-Golgi network (TGN, 20 degrees C). To localize the G protein specifically to ER exit sites, we incubated cells at 10 degrees C. The exit sites contained Sec13p, a COPII component, and were devoid of calnexin and other ER chaperones. We found that if the G protein in the exit sites was misfolded by a temperature shift from 10 degrees C to 39.5 degrees C, it failed to enter the VTCs. Instead, it was returned to the reticular ER where it associated with calnexin. However, if the G protein was in the VTCs or beyond, its folding status no longer affected further transport. The observations indicate that quality control took place in the ER and in the ER transitional elements, but not in the VTCs or the Golgi complex. The results provide a way to discriminate biochemically between exit sites and VTCs, two related structures that are difficult to distinguish from each other.