All vertebrate cells activate Cl- currents (I-Cl(,swell)) when swollen by hypotonic bath solution. The volume-regulated anion channel VRAC has now been identified as LRRC8/SWELL1. However, apart from VRAC, the Ca2+-activated Cl- channel (CaCC) TMEM16A and the phospholipid scramblase and ion channel TMEM16F were suggested to contribute to cell swelling-activated whole-cell currents. Cell swelling was shown to induce Ca2+ release from the endoplasmic reticulum and to cause subsequent Ca2+ influx. It is suggested that TMEM16A/F support intracellular Ca2+ signaling and thus Ca2+-dependent activation of VRAC. In the present study, we tried to clarify the contribution of TMEM16A to I-Cl(,swell). In HEK293 cells coexpressing LRRC8A and LRRC8C, we found that activation of I-Cl(,swell) by hypotonic bath solution (Hypo; 200 mosm/l) was Ca2+ dependent. TMEM16A augmented the activation of LRRC8A/C by enhancing swelling-induced local intracellular Ca2+ concentrations. In HT29 cells, knockdown of endogenous TMEM16A attenuated I-Cl(,swell) and changed time-independent swelling-activated currents to VRAC-typical time-dependent currents. Activation of I-Cl(,swell) by Hypo was attenuated by blocking receptors for inositol trisphosphate and ryanodine (IP3R; RyR), as well as by inhibiting Ca2+ influx. The data suggest that TMEM16A contributes directly to I-Cl(,swell) as it is activated through swelling-induced Ca2+ increase. As activation of VRAC is shown to be Ca2+-dependent, TMEM16A augments VRAC currents by facilitating Hypo-induced Ca2+ increase in submembraneous signaling compartments by means of ER tethering.