To identify the ion channels involved in erythrocyte volume regulation, whole-cell currents from human red blood cells (RBCs) were recorded in isotonic, hypotonic and hypertonic media. In isotonic NaCl bath solution, whole-cell currents rectified outwardly with reversal potentials (Vrev) between the equilibrium potential of Cl- (ECl) and that of nonselective cations (NSC), ENSC. Replacement of bath Cl by gluconate decreased outward conductance (G outward) by 43 +/- 6% (n = 5) and shifted Vrev with ECl indicating a high fractional Cl- conductance. Hypotonic cell swelling reversibly decreased G outward by 23 +/- 3% (n = 8) while hypertonic cell shrinkage reversibly increased G outward by 27 +/- 8% (n = 5). These shrinkage-activated and swelling-inactivated current fractions rectified outwardly with Vrev at ENSC suggesting that both fractions are generated by the same type of NSC channel. The shrinkage-activated deltaG outward decreased from 4.7 +/- 1.2 nS (n = 3) to 1.4 +/- 0.4 nS (n = 5) and 0.5 +/- 0.4 nS (n = 7) with the increase of pipette [Cl-] from 7 mM to 39 mM and 139 mM, respectively. Similarly, with this increase of pipette [Cl-], G outward under isotonic control conditions decreased from 8.2 +/- 1.4 nS (n = 5) to 7.4 +/- 1.0 nS (n = 20) and 4.1 +/- 0.7 nS (n = 17), due to the differing activity of these NSC channels. In conclusion, human RBCs express, besides a high fractional Cl- conductance, NSC channels that are regulated by cell volume and the cytosolic [Cl-].