The UDP-glucose:glycoprotein glucosyltransferase (GT) is a protein folding sensor and glycosyltransferase that constitutes an important component of the protein quality control machinery. With the use of quantitative immunogold electron microscopy, we established the subcellular distribution of GT in rat liver and pancreas and Drosophila melanogaster salivary gland as well as cell lines and correlated it with that of glucosidase II, calreticulin, and pre-Golgi intermediate markers. Labeling for GT, as well as for glucosidase II and calreticulin, was found in the endoplasmic reticulum (ER), including nuclear envelope and pre-Golgi intermediates located between ER and Golgi apparatus, and in the cell periphery. In the rough ER, labeling for GT was inhomogeneous, with variously sized labeled and unlabeled cisternal regions alternating, indicative of a meshwork of quality control checkpoints. Notably, labeling intensity for GT was highest in pre-Golgi intermediates, corresponding to twice that of rough ER, whereas the Golgi apparatus exhibited no specific labeling. These results suggest that protein quality control is not restricted to the ER and that the pre-Golgi intermediates, by virtue of the presence of GT, glucosidase II, and calreticulin, are involved in this fundamental cellular process.