Early gentamicin nephrotoxicity is characterized by a variety of renal transport abnormalities, including polyuria secondary to nephrogenic diabetes insipidus. To investigate whether gentamicin directly interferes with cellular mechanisms responsible for ADH responsivity as a possible contributing factor in this tubular transport alteration, the effect of gentamicin on ADH-stimulated water flow was examined in vitro in the toad urinary bladder. Gentamicin decreased ADH-stimulated osmotic water flow in a dose-dependent manner, with a threshold effect at approximately 0.5 mM. This inhibitory effect occurred only in response to submaximal concentrations of ADH, was reversible, and was preventable with Mg++. Additionally, gentamicin reduced theophylline-stimulated osmotic water flow but had no effect on cyclic AMP-induced water flow. These effects are consistent with a direct gentamicin-related decrease in ADH-responsive adenylate cyclase activity and suggest a mechanism that may contribute to the nephrogenic diabetes insipidus characteristic of early gentamicin nephrotoxicity and the nonoliguric acute renal failure that is a late toxic event occurring with the use of this antibiotic.