Inhibition of the Na conductance of the apical membrane of the toad urinary bladder by amiloride, alkali cations and protons was voltage dependent. Bladders were bathed with a high K-sucrose serosal medium to reduce series basal-lateral resistance and potential difference. Transepithelial current-voltage relationships were measured over a voltage range of +/- 200 mV with a voltage ramp of frequency 0.5 to 1 Hz. Na channel I-V relationships were obtained by subtraction of currents measured in the presence of maximal doses of amiloride (10 to 20 microM). With submaximal doses of amiloride (0.05 to 0.5 microM), the degree of inhibition of the Na channel current (INa) increased as the mucosal potential was made more positive. The data can be reasonably well explained by assuming that amiloride blocks Na transport by binding to a site which senses approximately 12% of the transmembrane voltage difference. INa was reduced in a qualitatively similar voltage-dependent manner by mucosal K, Rb, Cs and Tl (approximately 100 mM) and by mucosal H (approximately 1 mM). Block by these cations cannot be explained in terms of interactions with a single membrane-voltage-sensing site; a model in which there are two or more blocking sites in series provides a better description of the data. On the other hand, amiloride block was reduced competitively by mucosal Na and K, suggesting that occupation of the channel by one cation excludes occupancy by the others. ADH and ouabain also reduce the apparent affinity of amiloride for its blocking site. Thus, intracellular Na may also compete with amiloride for occupancy of the channel.