The effect of the K(+)-sparing diuretic amiloride and two of its hydrophobic analogs, methylisobutyl amiloride (MIA) and ethylisopropyl amiloride (EIPA), on Ca-activated K+ channels from renal microvillus membrane vesicles incorporated into planar lipid bilayers was investigated. Amiloride did not inhibit currents through Ca-activated K+ channels. MIA and EIPA, however, inhibited channel currents when added to both the internal and external solutions in concentrations between 10 and 250 microM. Furthermore, when dose-response data for channel inhibition were examined using Hill plots, Hill numbers of approximately 1.5 were found for both blockers from both sides, suggesting that the mechanism of block involves multiple inhibitory binding sites. A simple kinetic scheme is proposed that can account for the results.