The G-protein activators guanosine 5'-O-(3-thiodiphosphate) (GTP gamma S) and aluminum fluoride (AIF) are thought to inhibit transport between Golgi cisternae by causing the accumulation of nonfunctional coatomer-coated transport vesicles on the Golgi. Although GTP gamma S and AIF inhibit transport in cell-free intra-Golgi transport systems, blocking coatomer vesicle formation does not. We therefore determined whether inhibition of in vitro Golgi transport by these agents requires coatomer vesicle formation. Depletion of coatomer was found to completely block coated vesicle formation on Golgi cisternae without affecting inhibition of in vitro transport by either GTP gamma S or AIF. Depletion of ADP-ribosylation factor (ARF) prevented inhibition of transport by GTP gamma S, but not by AIF, suggesting that the AIF-sensitive component in transport may not be a GTP-binding protein. Surprisingly, depletion of cytosolic ARF did not prevent the GTP gamma S-induced formation of Golgi-coated vesicles, whereas ARF was required for AIF-induced vesicle formation. Although ARF or coatomer depletion caused an increase in the fenestration of cisternae, no other ultrastructural changes were observed that might explain the inhibition of transport by GTP gamma S or AIF. These findings suggest that ARF-GTP gamma S and AIF act by distinct and coatomer-independent mechanisms to inhibit membrane fusion in cell-free intra-Golgi transport.