Cells of the renal inner medullary collecting duct (IMCD) are able to regulate their volume after hypoosmotic perturbations (regulatory volume decrease, RVD) by activating a swelling-sensitive anion/organic osmolyte channel (VRAC) [1]. As full activation of VRAC takes several minutes it might be of advantage for the IMCD cells to recruit other anion conductances for support, especially in case of massive hypoosmotic stress. A possible candidate might be the Ca²⁺-activated Cl^– conductance (CaCC), as it can be recruited rapidly by even a small increase in intracellular calcium ([Ca²⁺]_in) [2]. To investigate this hypothesis, cells isolated from the initial third of the mouse IMCD (mIMCD; see [2]) were cultured on glass coverslips (Media osmolality: 600 mosmol/kg H₂O) and challenged with either a moderate (bath osmolality reduction by 100 mosmol/kg H₂O via removal of sucrose) or a massive (bath osmolality reduction by 300 mosmol/kg H₂O via removal of sucrose) hypoosmotic shock, respectively. Membrane conductance changes of the cells were investigated using the slow whole cell patch-clamp technique (Nystatin perforated patch). Currents were attributed to VRAC or CaCC by their distinct biophysical characteristics (see [1]). Alterations in [Ca²⁺]_in were measured by intracellular ratiometric Ca²⁺ imaging using the fluorescent calcium indicator Fura2. Data are given as mean±SEM (n), statistical significance was tested as in [1]. A moderate hypoosmotic challenge via a reduction of the extracellular osmolality by 100 mosmol/kg H₂O resulted in activation of VRAC. However, neither a significant increase in [Ca²⁺]_in nor an activation of CaCC was detectable. On the other hand, challenging the mIMCD cells with a massive hypoosmotic shock (-300 mosmol/kg H₂O) elicited not only VRAC activity but also an increase in [Ca²⁺]_in in conjunction with activation of CaCC. [Ca²⁺]_in rose from a basal level of 28±5nM (n=7) to 367±21nM (n=7), and membrane conductance grew from -2.4±0.8pA/pF to -12.2±1.2pA/pF at -80mV and from 4.2±0.9pA/pF to 72±2.1pA/pF at +80mV (n=11), respectively. The increase in [Ca²⁺]_in could be detected 31±5s (n=10) after the start of the hypoosmotic challenge and lasted as long as the osmotic gradient was maintained. Omission of calcium in the extracellular bathing solution ([Ca²⁺]_ex < 10nM) had neither a significant effect on the intracellular Ca²⁺ signal nor on the CaCC activity elicited by the hypoosmotic challenge. Chelation of intracellular Ca²⁺ on the other hand prevented the [Ca²⁺]in rise as well as CaCC activation. This indicates intracellular calcium stores as the main Ca²⁺ source for the signal. In conclusion, activation of CaCC via an intracellular calcium signal seems to support the RVD of mIMCD cells after a strong hypoosmotic challenge by providing an early anion efflux pathway which precedes the slower activating VRAC conductance.