Gramicidin A forms univalent cation-selective channels of approximately 4 A diameter in phospholipid bilayer membranes. The transport of ions and water throughout most of the channel length is by a single-file process; that is, cations and water molecules cannot pass each other within the channel. The implications of this single-file mode of transport for ion movement are considered. In particular, we show that there is no significant electrostatic barrier to ion movement between the energy wells at the two ends of the channel. The rate of ion translocation (e.g., Na+ or Cs+) through the channel between these wells is limited by the necessity for an ion to move six water molecules in single file along with it; this also limits the maximum possible value for channel conductance. At all attainable concentrations of NaCl, the gramicidin A channel never contains more than one sodium ion, whereas even at 0.1 M CsCl, some channels contain two cesium ions. There is no necessity to postulate more than two ion-binding sites in the channel or occupancy of the channel by more than two ions at any time.