Apoptosis is characterized by a conserved series of morphological events beginning with the apoptotic volume decrease (AVD). This study investigated a role for aquaporins (AQPs) during the AVD. Inhibition of AQPs blocked the AVD in ovarian granulosa cells undergoing growth factor withdrawal and blocked downstream apoptotic events such as cell shrinkage, changes in the mitochondrial membrane potential, DNA degradation and caspase-3 activation. The effects of AQP inhibition on the AVD and DNA degradation were consistent in thymocytes and with two additional apoptotic signals, thapsigargin and C6-ceramide. Overexpression of AQP 1 in Chinese Hamster Ovary cells (CHO-AQP 1 cells) enhanced their rate of apoptosis. The AVD is driven by loss of K⁺ from the cell and we hypothesize that following the AVD, AQPs become inactive, halting further water loss and allowing K⁺ concentrations to decrease to levels necessary for apoptotic enzyme activation. Swelling assays on granulosa cells, thymocytes and CHO-AQP1 cells revealed that indeed the shrunken (apoptotic) subpopulation has a very low water permeability compared to the normal sized (nonapoptotic) subpopulation. In thymocytes, AQP1 is present and was shown to co-localize with a plasma membrane receptor, TNF-R1, both before and after the AVD, suggesting that this protein is not proteolytically cleaved and remains on the cell membrane. Overall, these data indicate that AQP-mediated water loss is important for the AVD and downstream apoptotic events, that the water permeability of the plasma membrane can control the rate of apoptosis and that inactivation following the AVD may help create the low K⁺ concentration essential in apoptotic cells. Furthermore, inactivation of AQPs after the AVD does not appear to be through degradation or removal from the cell membrane.