This study describes a precise and sensitive method for the bioassay of 8-arginine vasotocin (AVT). The technique is based on the observation that AVT increases the permeability to urea of the isolated toad bladder and that this effect can be measured with minute quantities of AVT by suspending bladders in humid air and applying hormone on small paper disks. The hormone diffuses from the disk to the underlying epithelium where it increases permeability to urea. The bladder is filled with Ringer's fluid containing [14C]urea and the label diffuses across the bladder wall into the disk at the serosal surface. Disks are removed at timed intervals for scintillation counting. Disks containing 0.25 pg AVT accumulated 232 +/- 8 cpm of [14C]urea in 30 min, whereas control disks without hormone at a distance of 3-4 mm on the same bladder wall accumulated only 12 +/- 2 cpm. The minimal sensitivity during the summer was 0.1 pg AVT. Average values (+/- SE) for induction of a half-maximal urea permeability response in toads from all seasons were 1.4 +/- 0.3 X 10(-10) M for AVT, 7.4 +/- 1.5 X 10(-9) M for 8-arginine vasopressin, and 2.9 +/- 0.6 X 10(-8) M for oxytocin. Plasma from a hydrated toad (255 mosm/kg H2O) showed AVT-like activity of 1.53 X 10(-11) M and from a dehydrated toad (342 mosm/kg H2O) 4.86 X 10(-11) M. Because this bioassay is specific and as sensitive as the radioimmunoassay, it may serve as a useful tool for studying the physiology of vasotocin.