Recently we reported a simple manual assay for the measurements of isotope fluxes through channels in heterogenous vesicle populations (Garty et al.,J. Biol. Chem. 258:13094–13099 (1983)). The present paper describes the application of this method to the assessment of amiloride blockable fluxes in toad bladder microsomes. When²²Na⁺ uptake was monitored in the presence of an opposing Na⁺ gradient, a relatively large and transient amiloride-sensitive flux was observed. Such an amiloride-blockable flux could also be induced by a KCl+valinomycin diffusion potential. The effects of the intra- and extravesicular ionic composition on the rate of²²Na⁺ uptake were examined. It was shown that the amiloride-blockable fluxes occur in particles permeable to Na⁺ and Li⁺ but relatively impermeable to K⁺, Tris⁺ and Cl^–. Analysis of the amiloride dose-response relations revealed a complex non Michaelis-Menten behavior. The data could be accounted for by assuming either a strong negative cooperativity in the amiloride-membrane interaction, or two amiloride-sensitive Na⁺ conducting pathways withK _i values of 0.06 and 6.4 m. Both pathways appear to be electrogenic and therefore the possibility of an electroneutral amiloride-blockable Na/H exchange was excluded. Calcium ions could block the amiloride-sensitive flux from the inner but not from the outer phase of the membrane. It is suggested that although a substantial part of the²²Na⁺ flux is inhibited only by a relatively high concentration of amiloride, this uptake represents transport through the apical Na-specific channels. The data also define the optimal experimental conditions for the study of amiloride-sensitive fluxes in toad bladder microsomes.