A regulatory volume decrease is accomplished by parallel activation of Ca(2+)-dependent K+ channels and Ca(2+)-independent Cl- channels in cultured human intestinal epithelial cells (Intestine 407). The anion selectivity of whole-cell currents recorded in osmotically swollen cells falls into the Eisenman type I sequence corresponding to a low-field anion channel. The volume-sensitive Cl- channel has an intermediate unitary conductance. Both the whole-cell and single-channel Cl- currents exhibit unique voltage-dependency. The Cl- current can be maintained in the activated state in the physiological voltage range. However, at very large depolarizations (over +50 mV), the current is quickly inactivated. The Cl- current shows moderate outward rectification. The whole-cell Cl- current is sensitive to Cl- channel blockers such as SITS and NPPB as well as to cis unsaturated fatty acids such as arachidonic acid and oleic acid. The whole-cell current is totally independent of Ca2+ and cyclic AMP, but inhibited by increases in cytosolic free Mg2+ ions. Removal of intracellular ATP, but not Mg2+, abolishes the Cl- current. The ATP role can be substituted for non-hydrolyzable ATP analogs. Therefore, it is likely that intracellular ATP maintains the channel activity through non-hydrolytic binding.