Previously, we demonstrated that β1,4galactosyltransferase (gal-T1) reversibly segregates from α2,6sialyltransferase (ST6Gal) to swollen vesicles after monensin treatment of the cells. To further explore this phenomenon, we investigated the response to monensin of various Golgi proteins. Within 30 min of monensin treatment, gal-T1 moved from the Golgi apparatus, as defined by localization of giantin, to swollen vesicles whereas ST6Gal, α2,3(N)sialyltransferase, mannosidase II, and N-acetylgalactosaminyltransferase 2 remained associated with the Golgi apparatus. Stably transfected CHO cells exhibited a similar phenomenon of monensin-induced displacement of recombinant gal-T1 to swollen vesicles while recombinant ST6Gal remained colocalized with endogenously expressed giantin. Gal-T1 and the cation-insensitive mannose 6-phosphate receptor colocalized in swollen vesicles as observed at both light and electron microscopic levels. When monensin was replaced by chloroquine, gal-T1 remained arrested in swollen vesicles. Brefeldin A treatment known to cause relocation of Golgi-associated gal-T1 to the endoplasmic reticulum had no effect on gal-T1 trapped in swollen vesicles. This evidence suggests that monensin blocks gal-T1 trafficking in post-Golgi structures and argues against swelling of gal-T1-containing trans Golgi cisternae as previously assumed.