The small GTP-binding protein Rab6, a member of the Ras superfamily, is localized on the membranes of the Golgi apparatus and the trans Golgi network. Recent studies revealed that the Rab6 protein might be involved in the transit of proteins through the Golgi complex. In this report we demonstrate the essential function of the Rab6 protein in a distinct step of reconstituted Golgi transport. Polyclonal antibodies and Fab fragments directed against the C-terminal part of the Rab6 protein inhibit transport between the cis and the medial Golgi cisternae. Inhibition also occurred when a trans-dominant mutant form of the Rab6 protein (N126I) was added to the reconstituted transport. Furthermore, Rab6 antibodies inhibit uncoupled fusion of Golgi membranes. From these data we conclude that Rab6 is involved in a process related to membrane fusion at the cisternal membranes of the Golgi apparatus and therefore is needed for the consumption of Golgi-derived vesicles by their target membranes.