A major focus for our laboratory is identifying the molecules and mechanisms that regulate basolateral-to-apical transcytosis in polarized hepatocytes. Our most recent studies have focused on characterizing the biochemical and functional properties of the small rab17 GTPase. We determined that rab17 is a monosumoylated protein and that this modification likely mediates selective interactions with the apically located syntaxin 2. Using polarized hepatic WIF-B cells exogenously expressing wild-type, dominant active/guanosine triphosphate (GTP)-bound, dominant negative/guanosine diphosphate (GDP)-bound, or sumoylation-deficient/K68R rab17 proteins, we confirmed that rab17 regulates basolateral-to-apical transcytotic vesicle docking and fusion with the apical surface. We further confirmed that transcytosis is impaired from the subapical compartment to the apical surface and that GTP-bound and sumoylated rab17 are likely required for apical vesicle docking. Because expression of the GTP-bound rab17 led to impaired transcytosis, whereas wild type had no effect, we further propose that rab17 GTP hydrolysis is required for vesicle delivery. We also determined that transcytosis of three classes of newly synthesized apical residents showed similar responses to rab17 mutant expression, indicating that rab17 is a general component of the transcytotic machinery required for apically destined vesicle docking and fusion.