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. When 22Na+ 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 of 22Na+ 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 with Ki values of 0.06 and 6.4 microM. 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 the 22Na+ 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.