The diuretic compound amiloride is often used as a specific inhibitor of the passive Na+ entry step in the transepithelial transport of Na+ across frog skin. We have utilized the fluorescence properties of amiloride to study the distribution of this transport inhibitor in the ventral skin of Rana pipiens. After a 30 s exposure of 1-100 microM amiloride to the external surface of frog skin, amiloride fluorescence was evident in the cytoplasm of all cell layers of the epidermis and alveolar gland epithelium. Changes in the conditions of incubation which alter the pharmacological activity of amiloride did not affect the intracellular distribution of amiloride or the washout profile of [14C]amiloride. The presence of amiloride fluorescence in the cytoplasm prevented our examination of changes in the amiloride fluorescence at the cell surface with various conditions of incubation. Four derivatives of amiloride that differed in their ability to inhibit short-circuit current were also localized intracellularly but varied in their relative distribution among the cell layers of the epidermis. Our results indicate that when incubated at concentrations from 1 to 100 microM, a large fraction of the amiloride taken up by frog skin is not directly involved with the inhibition of passive Na+ transport at the apical surface of the stratum granulosum. The mechanism of intracellular uptake of amiloride is not clear. However, the cytoplasmic localization of amiloride could explain the action of the drug on intracellular enzymes and may account for the large proportion of non-displaceable [14C]amiloride that has been observed in frog skin.