Vasopressin-stimulated cyclic AMP content and the uptake of Hz 0 and 22 Na into canine renal medullary slices were measured. Cyclic AMP was increased threefold by 9 X lO-'M vasopressin in isotonic (290 mOsm/kg HzO) Krebs-Ringers bicarbonate. A significant increase in vasopressin-stimulated Hz 0 uptake began at 2.75 min after hormone addition and lasted until 5.00 min. Colchicine (1 X lo-' M) inhibited the vasopressin-stimulated Hz 0 uptake. This effect required a minimum preincubation period of 30-40 min in colchicine-containing medium. Colchicine had n o effect on basal or vasopressin-stimulated cyclic AMP levels. Colchicine also inhibited had no effect on either vasopressin-or dibutyryl cyclic AMP-stimulated uptake. l4 C-colchicine bound predominantly t o the cytosol fraction enriched in microtubules, while virtually n o binding was observed on plasma membranes. Light-microscopic examinations of cross sections of tissue slices showed that a majority of vasopressin-treated collecting tubules and some control tubules had occluded lumens. Colchicine-treated cells, in the presence of vasopressin, had open lumens indicating a blockage of the vasopressin-induced water transport. Cells treated with cytochalasin B (1 pgm/ml) also had open lumens in the presence of vasopressin. Cytochalasin B also blocked vasopressin and dibutyryl cyclic AMPstimulated H2 0 uptake into collecting duct cells but had n o effect on vasopressinstimulated cyclic AMP levels. It was concluded that microtubules and possibly microfilaments are involved in the subcellular mechanism by which vasopressin increases the permeability of the collecting duct to water. Hz 0 uptake stimulated by dibutyryl cyclic AMP ( 10 mM). Lumicolchicine (1 O r 5 M) Hz 0