Xenopus oocytes are frequently utilized for in vivo expression of cellular proteins, especially ion channel proteins. A thorough understanding of the endogenous conductances and their regulation is paramount for proper characterization of expressed channel proteins. Here we detail a novel chloride current (Icl.swell) responsive to hypotonicity in Xenopus oocytes using the two-electrode voltage clamp technique. Reducing the extracellular osmolarity by 50% elicited a calcium-independent chloride current having an anion conductivity sequence identical with swelling-induced chloride currents observed in epithelial cells. The hypotonicity-activated current was blocked by chloride channel blockers, trivalent lanthanides, and nucleotides. G-protein, cAMP-PKA, and arachidonic acid signaling cascades were not involved in Icl.swell activation. Icl.swell I is distinct from both stretch-activated nonselective cation channels and the calcium-activated chloride current in oocytes and may play a critical role in volume regulation in Xenopus oocytes.