I C l n , a cytosolic protein associated with a nucleotide-sensitive chloride current, may be involved in the regulation of a volume-regulated anion current (VRAC) associated with hypotonic cell swelling. We have determined the nucleic acid sequences of I C l n from human tsA201a, colonic (T84) and myeloma (RPMI 8826) cell lines. The amino acid sequences are highly homologous (=<99%) to each other but less homologous to I C l n protein from other species. Using the whole-cell patch clamp technique, we examined the effect of I C l n protein expression levels on VRAC properties during a hyposmotic challenge. Overexpression of T84 or RPMI 8226-derived I C l n protein in tsA201a cells results in a more than 9-fold increase in the rate of VRAC activation over control values, while having no effect on VRAC inactivation properties. Underexpression of endogenous I C l n protein in tsA201a cells using antisense oligonucleotides results in a more than 180-fold decrease in VRAC activation rate as compared to control values. These results indicate that I C l n protein expression modulates VRAC activation but not inactivation.