1. The effect of the dynein inhibitor erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA) on the osmotic water flow response to vasopressin or exogenous cAMP has been investigated in isolated toad urinary bladders. 2. Pretreatment with serosal EHNA had no effect on basal water flow, but inhibited the development and maintenance of the hydrosmotic response to vasopressin (20 mU ml-1) or 8-(4-parachlorophenylthio)-adenosine 3',5'-cyclic monophosphate (8 CPT-cAMP; 0.1 mM). 3. The inhibitory effect of EHNA on vasopressin-induced water flow was dose dependent. Inhibition occurred in the dose range in which EHNA inhibits the ATPase and motor activities of dynein in vitro. 4. EHNA also inhibited the maintenance of the high rate of water flow established by prior exposure to vasopressin. 5. The inhibitory effect of EHNA on the onset phase of the vasopressin response was attenuated after exposure of the tissue to the microtubule-disruptive drug nocodazole but was fully additive with that of cytochalasin B. 6. EHNA inhibited basal and vasopressin-stimulated transepithelial sodium transport. 7. The findings support the view that EHNA inhibits hormone-induced water flow through an action on a cytoplasmic dynein. The results are consistent with the hypothesis that dynein is involved in the microtubule-based delivery of water channel-containing vesicles to the apical membrane of the granular epithelial cells during both the onset and maintenance of the water permeability response to vasopressin.