We studied the effects of hypertonic stress on ion transport and cell volume regulation (regulatory volume increase; RVI) in the human tumor cell-line HepG2. Ion conductances were monitored in intracellular current-clamp measurements with rapid ion-substitutions and in whole-cell patch-clamp recordings; intracellular pH buffering capacity and activation of Na+/H+ antiport were determined fluorometrically; the rates of Na+-K(+)2Cl- symport and Na+/K+-ATPase were quantified on the basis of time-dependent and furosemide- or ouabain- sensitive Rb-86(+) uptake, respectively; changes in cell volume were recorded by means of confocal laser-scanning microscopy. It was found that hypertonic conditions led to the activation of a cation conductance that was inhibited by Gd3+, flufenamate as well as amiloride, but not by benzamil or ethyl-isopropyl- amiloride (EIPA). Most likely, this cation conductance was non- selective for Na+ over K+. Hypertonic stress did not change K+ conductance, whereas possible changes in Cl- conductance remain ambiguous. The contribution of Na+/H+ antiport to the RVI process appeared to be minor. Under hypertonic conditions an approximately 3.5-fold stimulation of NaK+-2Cl- symport was observed but this transporter did not significantly contribute to the overall RVI process. Hypertonic stress did not increase the activity of Na+/K(+)ATPase, which even under isotonic conditions appeared to be working at its limit. It is concluded that the main mechanism in the RVI of HepG2 cells is the activation of a novel non-selective cation conductance. In contrast, there is little if any contribution of K+ conductance, Na+/H+ antiport, Na+-K+-2Cl- symport, and Na+/K+- ATPase to this process.