Brefeldin A (BFA) induces the retrograde transport of proteins from the Golgi complex (GC) to the endoplasmic reticulum (ER). It is uncertain, however, whether the drug completely merges the ER with post-ER compartments, or whether some of their elements remain physically and functionally distinct. We investigated this question by the use of monoclonal antibodies specific for monomers and trimers of the influenza virus hemagglutinin (HA). In untreated influenza virus-infected cells, monomers and trimers almost exclusively partition into the ER and GC, respectively. In BFA-treated cells, both monomers and trimers are detected in the ER by immunofluorescence. Cell fractionation experiments indicate, however, that whereas HA monomers synthesized in the presence of BFA reside predominantly in vesicles with a characteristic density of the ER, HA trimers are primarily located in lighter vesicles characteristic of post-ER compartments. Biochemical experiments confirm that in BFA-treated cells, trimers are more extensively modified than monomers by GC-associated enzymes. Additional immunofluorescence experiments reveal that in BFA-treated cells, HA monomers can exist in an ER subcompartment less accessible to trimers and, conversely, that trimers are present in a vesicular compartment less accessible to monomers. These findings favor the existence of a post-ER compartment for which communication with the ER is maintained in the presence of BFA and suggest that trimers cycle between this compartment and the ER, but have access to only a portion of the ER.