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The effect of the K-sparing diuretic amiloride was assessed electrophysiologically in the isolated cortical collecting tubule of the rabbit, a segment which absorbs Na and secretes K. Low concentrations of amiloride in the perfusate caused a rapid, reversible, decrease in the magnitude of the lumen negative transepithelial potential difference, Vte, transepithelial conductance Gte, and equivalent short-circuit current, Isc, with an apparent K1/2 of approximately 7 X 10(-8) M. The effects of a maximum inhibitory concentration of amiloride (10(-5) M) were identical to those observed upon Na removal from lumen and bath (Na removal from the bath alone has no effect). Removal of Na in the presence of 10(-5) M amiloride had no affect on Vte, Gte, or Isc, and is consistent with the view that amiloride blocks the Na conductive pathways of the apical cell membrane. Further, in the absence of Na, the subsequent addition of amiloride had no influence. In tubules where active Na absorption was either spontaneously low, or abolished by removal of Na from lumen and bath, the elevation of K from 5 to 155 meq/liter in the perfusate caused a marked change of the Vte in the negative direction and an increase in the Gte. These effects could be attributed to a high K permeability of the apical cell membrane and not of the tight junctions. Amiloride (10(-5) M) had no effect on these responses to K. It is concluded that amiloride selectively blocks the apical cell membrane Na channels but has no effect on the K conductive pathway(s). This selective nature of amiloride may indicate that Na and K are transported across the apical cell membrane via separate conductive pathways.