Cells maintain control of their volume by the passage of KCl and water across their membranes, but the regulatory proteins are unknown. Expression in Xenopus oocytes of a novel protein, pICln, activated a chloride conductance. We have cloned analogs of pICln from rat heart and Xenopus ovary. pICln was identified as an abundant soluble cytosolic protein (approximately 40 kd) that does not immunolocalize with the plasma membrane. pICln was found in epithelial and cardiac cells, brain, and Xenopus oocytes, forming complexes with soluble actin and other cytosolic proteins. Monoclonal antibodies recognizing pICln blocked activation of a native hypotonicity-induced chloride conductance (ICl.swell) in Xenopus oocytes, suggesting that pICln may link actin-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel. The high degree of sequence conservation and widespread expression of pICln suggest that it is an important element in cellular volume regulation.