Amiloride analogues with N5alkyl substitutions are specific high-affinity ligands for the Na+-H+exchanger in various tissues. As a means to characterize the Na+-H+exchanger in the bovine corneal epithelium, we determined the binding properties of [3H] methylisobutylamiloride (MIA) to a fraction enriched in plasma membrane from this tissue. [H]MIA bound to these membranes in a time,-a temperature-, and -a pH-dependent manner. The binding was optimal at 4°C and at pH 8.5 and it reached equilibrium at 60 min. Under these conditions, specific binding, which was inhibitable by excess unlabeled MIA, was about 85%. Scatchard analysis of this specific binding revealed a single saturable binding component with a Kdof 61 nM and a Bmaxof 271 pmoles/mg protein. Inhibition of [3H]MIA specific binding by amiloride analogues showed the following order of potency: MIA>dimethylamiloride (DMA)>benzamil>amiloride. Na+did not compete with MIA for binding. The effectiveness of clonidine, an alpha2agonist, and cimetidine, an H2receptor antagonist, as inhibitors of Na+-H+exchange activity was also determined because these compounds are used to distinguish between the exchanger subtypes. At concentrations higher than those needed for receptor interaction, clonidine was more effective than cimetidine in decreasing MIA binding.The activity of Na+-H+exchanger, which was measured as the uptake of Na+in the presence of an outwardly directly H+gradient, was also inhibited by DMA, benzamil and amiloride with the same order of potency as obtained in the binding studies. As seen with the MIA binding studies, clonidine was more potent than cimetidine in inhibiting 22+Nauptake. We conclude that the specific binding of MIA observed in these membrane preparations represents the binding of the ligand to the Na+-H+exchanger and that the MIA binding site on the exchanger is distinct from the Na+binding site (substrate site). The relative potencies of clonidine and cimetidine to inhibit MIA binding and22Na+uptake indicate that the Na+-H+exchanger present in the corneal epithelial plasma membrane belongs to the amiloride-insensitive type that has been described in the brush-border membrane of the kidney and intestine.