Brefeldin A (BFA) is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. At low concentrations (5-10 microg ml(-1)), BFA caused both the Golgi apparatus and trans-Golgi network (TGN), an early endosome (EE) equivalent in plant cells, to form visible aggregates in transgenic tobacco BY-2 cells. Here we show that these BFA-induced aggregates from the Golgi apparatus and TGN are morphologically and functionally distinct in plant cells. Confocal immunofluorescent and immunogold electron microscope (EM) studies demonstrated that BFA-induced Golgi- and TGN-derived aggregates are physically distinct from each other. In addition, the internalized endosomal marker FM4-64 co-localized with the TGN-derived aggregates but not with the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, which acts in a dose- and time-dependent manner, SCAMP1 (secretory carrier membrane protein 1) and FM4-64 are mostly excluded from the SYP61-positive BFA-induced TGN aggregates, indicating that homotypic fusion of the TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE-derived BFA-induced aggregates. As the TGN also serves as an EE, continuously receiving materials from the plasma membrane, our data support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in terms of its response to BFA treatment in plant cells. Thus, the Golgi and TGN are probably functionally distinct organelles in plants.