We used an in vitro system based on streptolysin O-permeabilized MDCK cells to study the involvement of NSF, SNAP, SNAREs, and Rab proteins in polarized membrane transport of epithelial cells. In MDCK cells, transport from the trans-Golgi network (TGN) to the basolateral plasma membrane is inhibited by anti-NSF antibodies and stimulated by alpha-SNAP. In contrast, transport from the TGN to the apical cell surface is not affected by anti-NSF antibodies or alpha-SNAP. Furthermore, apical transport is insensitive to Rab-GDI and tetanus and botulinum neurotoxins, which inhibit basolateral transport. These results provide evidence that the Rab-NSF-SNAP-SNARE mechanism operates in basolateral transport, while other molecules constitute the machinery for vesicular delivery in the apical pathway.