Whilst the function and organization of the secretory machinery in eukaryotic cells exhibit basic similarities, the compartmentation of the endomembrane system can show significant differences between the fungal, plant and animal kingdoms. The use of the antibiotic brefeldin A (BFA) as an inhibitor of secretion in both animal and yeast cells has resulted in a remarkable advance in our understanding of the modes of action of vesicle shuttles between the endoplasmic reticulum and Golgi apparatus and within the Golgi apparatus itself. It is now apparent that application of the drug to filamentous fungi and plants will also help unravel the workings of the secretory system in these organisms. In this paper we review recent progress in our laboratories on elucidating the effects of BFA on the morphology of the Golgi apparatus and compare these with recently published data on fungal and plant cells. Variation in the response to BFA are reported, which may not all be attributed to differences in drug concentration and time of treatment. These may reflect differences in cellular sensitivity or multiple sites of action of the drug, and the existence of a specific molecular target for BFA is questioned.