The effects of the addition of graded concentrations of amiloride, (A)m, to the mucosal bathing solution on the permeability of the apical membrane of rabbit descending colon to Na (PmNa) were determined when the Na activity in the mucosal bathing solution, (Na)m, was 18, 32 or 100 mM. PmNa was obtained from current-voltage relations determined on tissues bathed with a high-K serosal solution before and after the addition of a maximally inhibitory concentration of amiloride to the mucosal solution as described by Turnheim et al. (Turnheim, K., Thompson, S.M., Schultz, S.G. 1983. J. Membrane Biol. 76:299-309). The results indicate that: (1) As demonstrated previously (Turnheim et al., 1983), PmNa decreases with increasing (Na)m. (2) PmNa also decreases hyperbolically with increasing (A)m. Kinetic analyses of the effect of amiloride on PmNa are consistent with the conclusions that: (i) the stoichiometry between the interaction of amiloride with apical membrane receptors that results in a decrease in PmNa is one-for-one; (ii) there is no evidence for cooperativity between amiloride and these binding sites; (iii) the value of (A)m needed to halve PmNa at a fixed (Na)m is 0.6-1.0 microM; and, (iv) this value is independent of (Na)m over the fivefold range studied. These findings are consistent with the notion that the sites with which amiloride interacts to bring about closure of the channels through which Na crosses the apical membrane are kinetically distinct from the sites with which (Na)m interacts to bring about closure (i.e., "self-inhibition"). In short, the effects of (Na)m and (A)m on PmNa in this tissue appear to be independent and additive.