The inhibition and stimulation of short-circuit current in in vitro frog skin by a series of structural analogs of the diuretic drug amiloride were studied. Also, the inhibitory profile of a new experimental diuretic, 3-(3-amino-1,2,4-oxadiazol-5-yl)-5-chloro-2,6-pyrazinediamine, or CGS 4270, was determined. The major conclusions of our studies are: 1) amiloride remains the most effective inhibitor of Na+ entry, with both the pyrazine ring and the acylguanidine moiety being required for maximum activity; 2) CGS 4270 also inhibits Na+ transport in frog skin; it acts only from the external solution, is uncharged, is noncompetitive with Na+ and interacts noncompetitively with amiloride; the inhibition is independent of external calcium; and 3) benzimidazole increases amiloride-sensitive short-circuit current and this compound is competitive with amiloride. These results indicate that the putative Na+ entry protein contains multiple inhibitory sites and that compounds which stimulate Na+ entry may also bind at the same molecular locus as an inhibitor.