We have visualized the relationship between the endoplasmic reticulum (ER) and Golgi in leaf cells ofNicotiana clevelandiiby expression of two Golgi proteins fused to green fluorescent protein (GFP). A fusion of thetrans-membrane domain (signal anchor sequence) of a rat sialyl transferase to GFP was targeted to the Golgi stacks. A second construct that expressed theArabidopsisH/KDEL receptor homologue aERD2, fused to GFP, was targeted to both the Golgi apparatus and ER, allowing the relationship between these two organelles to be studied in living cells for the first time. The Golgi stacks were shown to move rapidly and extensively along the polygonal cortical ER network of leaf epidermal cells, without departing from the ER tubules. Co-localization of F-actin in the GFP-expressing cells revealed an underlying actin cytoskeleton that matched precisely the architecture of the ER network, while treatment of cells with the inhibitors cytochalasin D and N-ethylmaleimide revealed the dependency of Golgi movement on actin cables. These observations suggest that the leaf Golgi complex functions as a motile system of actin-directed stacks whose function is to pick up products from a relatively stationary ER system. Also, we demonstrate for the first timein vivobrefeldin A-induced retrograde transport of Golgi membrane protein to the ER.