Colchicine and other antimitotic agents have been found to inhibit the action of vasopressin and cyclic AMP on transcellular water movement in the toad bladder; functional and biochemical studies suggest that the effect of these agents is due to interference with microtubule function. To further assess this hypothesis, a quantitative ultrastructural analysis of the content and distribution of microtubules was performed on epithelial cells of bladders exposed to colchicine, vasopressin, and cyclic AMP. The content (volume density) of microtubules was estimated by a point-counting stereological technique. The results indicate that the content of assembled microtubules in the granular epithelial cells is reduced in hemibladders exposed to colchicine; this effect is dose-dependent and is estimated to be half-maximal at a colchicine concentration of 1.4×10-6m. In contrast, the content of assembled microtubules in the granular cells is slightly (∼30%) but significantly increased after exposure of hemibladders to vasopressin (100 mU/ml) or cyclic AMP (10m m). The content of microtubules in mitochondria-rich cells was not found to be significantly altered after exposure to vasopressin. The combined results of functional, biochemical, and morphological studies provide evidence that cytoplasmic microtubules in the granular epithelial cells play a critical role in the action of vasopressin on transcellular water movement in the toad bladder. Precisely how microtubules are involved in the action of the hormone remains to be determined. © 1978 Springer-Verlag New York Inc.