A classical and stereotyped feature of apoptosis is cell shrinkage, also known as apoptotic volume decrease (AVD). AVD is driven by net loss of ions and osmotically obliged water and a prerequisite for caspase activation and execution of apoptosis. Here we show that cisplatin-induced AVD in Ehrlich ascites tumor cells (EATC) is a complex process consisting of at least 3 distinguishable stages defined by sequential efflux/influx of different ions, amino acids and water. These movements can be correlated to early and a late shrinkage stages and a transient cell volume recovery stage. While the shrinkage stages are associated with loss of K⁺, Cl^-, Na⁺ and amino acids, recovery is mediated by NaCl uptake. Cl^- efflux is a requisite component of AVD and, as expected, Cl^- channel inhibition in EATC alters AVD and NaCl movements, however in a unique manner in each AVD stage. To further investigate the importance and molecular mechanisms of Cl^- movements, we have cloned and expressed members of the recently identified TMEM16 family of Cl^- channels and analyzed their biophysical channel properties. When over expressed in HEK293 cells, mTMEM16F (mouse anoctamin 6) was found to induce an outwardly rectifying Ca²⁺-activated chloride current (I_Cl,Ca). The current activates time-dependently at high positive potentials, has an anion selectivity sequence of SCN^- > I^- > Br^- > Cl^- > Asp and is sensitive to tamoxifen > niflumic acid > NS3728 > DIDS. Mutation of R592, K616 and K636, the homelogue amino acids which in TMEM16A has been suggested to affect channel anion selectivity, to glutamic acid completely abolished the Ca²⁺ induced Cl^- current. In addition, we found that stable knock-down of mTMEM16F in EATC reduced I_Cl,Ca significantly. In conclusion, Cl^- movements are important during different stages of AVD and for the proper execution of apoptosis. mTMEM16F appears to be a CaCC which may play important roles.