We have previously reported in a rat cortical collecting duct cell line (RCCD₁) that the presence of aquaporin 2 (AQP2) in the cell membrane is critical for the rapid activation of regulatory volume decrease mechanisms (RVD) (Ford et al. Biol Cell 2005; 97: 687-697). The aim of our present work was to investigate the signaling pathway that links AQP2 to this rapid RVD activation. Since it has been previously described that hypotonic conditions induce intracellular calcium ([Ca²⁺]_i) increases in different cell types, we tested the hypothesis that AQP2 could have a role in activation of calcium entry by hypotonicity and its implication in cell volume regulation. Using a fluorescent probe technique, we have studied [Ca²⁺]_i and cell volume changes in response to a hypotonic shock in WT-RCCD₁ (not expressing aquaporins) and in AQP2-RCCD₁ (transfected with AQP2) cells. We found that after a hypotonic shock only AQP2-RCCD₁ cells exhibit a substantial increase in [Ca²⁺]_i . This [Ca²⁺]_i increase is strongly dependent on extracellular Ca²⁺ and is partially inhibited by thapsigargin (1 µM) indicating that the rise in [Ca²⁺]_i reflects both influx from the extracellular medium and release from intracellular stores. Exposure of AQP2-RCCD₁ cells to 100 µM gadolinium reduced the increase [Ca²⁺]_i suggesting the involvement of a mechanosensitive calcium channel. Furthermore, exposure of cells to all the above described conditions impaired rapid RVD. We conclude that the expression of AQP2 in the cell membrane is critical to produce the increase in [Ca²⁺]_i which is necessary to activate RVD in RCCD₁ cells.