Rotavirus is one of very few viruses that utilizes the endoplasmic reticulum (ER) for maturation. The maturation process is unique not only because it involves translocation of subviral particles across the ER membrane, but also because mature virus is thought to be retained in the ER until cell lysis. Brefeldin A (BFA) is a compound that blocks protein export from the ER to the Golgi complex and causes disruption of the Golgi complex with relocation of resident Golgi proteins to the ER. We found that BFA had a pronounced effect on rotavirus assembly and oligosaccharide processing. Single-step growth experiments demonstrated that BFA reduced infectious progeny rotavirus yield by 99.9%. Immunohistochemical staining with monoclonal antibodies showed that all examined VP4, VP6, VP7, and NS28 epitopes remained unaffected by BFA. A novel observation from pulse-chase experiments was that BFA-treatment rapidly increased the molecular weight of the ER-associated VP7 followed by endo-beta-N-acetylglucosaminidase H (endo H) resistance. A novel observation was also that the trans-ER NS28 protein remained endo H sensitive through the course of BFA-treatment, but that the molecular weight varied during chase. Electron microscopy analysis revealed that BFA interfered in the transition from the intermediate enveloped particle to the mature double-shelled virus.