Surface cells of the mammalian distal colon, which are involved in electrogenic Na⁺ absorption, are shown to molecularly express the amiloride-sensitive epithelial Na⁺ channel composed of three homologous subunits (-, -, and -ENaC). Since basic electrophysiological properties of amiloride-sensitive Na+ channels expressed in these cells are largely unknown at the cellular level, however, functional evidence for the involvement of the subunits in the native channels is incomplete. Using electrophysiological techniques, we have now characterized functional properties of native ENaC in surface cells of rectal colon (RC) of rats fed a normal Na⁺ diet. Ussing chamber experiments first showed that apical amiloride inhibited a basal short-circuit current (I_sc) in mucosal preparation of RC with an apparent half-inhibition constant (K_i) value of 0.20 µM. RT-PCR analysis confirmed the presence of transcripts of -, -, and -rENaC in rectal mucosa. Whole-cell patch-clamp experiments in surface cells of intact crypts acutely isolated from rectal mucosa identified an inward cationic current, which was reversibly inhibited by amiloride with a K_i value of 0.12 µM at a membrane potential of -64 mV. Amiloride block of the current was weakly voltage-dependent, yielding an electrical distance () of 0.24 sensed by the blocker. Conductance ratios of the currents were Li⁺ (1.8) > Na⁺ (1) >> K⁺ (~ 0), respectively. Amiloride-sensitive current amplitude was almost the same at 15 mM or 150 mM of extracellular Na⁺, suggesting a high Na+ affinity for current activation. These results collectively provide the first electrophysiological characterization of macroscopic currents mediated by amiloride-sensitive Na⁺ channels naturally expressed in surface cells of RC of rats and are consistent with the hypothesis that a heterooligomer composed of -, -, and -ENaC may be the molecular basis of the native channels, which are responsible for amiloride-sensitive electrogenic Na⁺ absorption in the mammalian distal colon.