ClC-3 is a Cl-/H+ antiporter required for intra-endosomal reactive oxygen species (ROS) generation by Nox1. ClC-3 current is distinct from the swelling activated chloride current (IClswell), but over-expression of ClC-3 can activate currents that resemble IClswell. Since H2O2 activates IClswell directly, we hypothesized that ClC-3-dependent, endosomal ROS production activates IClswell. Whole-cell perforated patch clamp methods were used to record Cl- currents in cultured aortic vascular smooth muscle cells from wild-type (WT) and ClC-3 NULL mice. Under isotonic conditions TNF-α (10 ng/ml) activated outwardly rectifying Cl- currents with time-dependent inactivation in WT, but not ClC-3 NULL cells. Inhibition by tamoxifen (10 μM) and by hypertonicity (340 mosm) identified them as IClswell. IClswell was also activated by H2O2 (500 μM) and the effect of TNF-α was completely inhibited by PEG-catalase. ClC-3 expression induced IClswell in ClC-3 NULL cells in the absence of swelling or TNF-α, and this effect was also blocked by catalase. IClswell activation by hypotonicity (240 mosm) was only partially inhibited by catalase and the size of these currents did not differ between WT and ClC-3 NULL cells. Disruption of endosome trafficking with either mutant Rab5 (S34N) or Rab11 (S25N) inhibited TNF-α-mediated activation of IClswell. Thrombin also activates ROS production by Nox1, but not in endosomes. Thrombin caused H2O2-dependent activation of IClswell, but this effect was not ClC-3 or Rab5-dependent. Thus, activation of IClswell by TNF-α requires ClC-3-dependent endosomal H2O2 production. This demonstrates a functional link between two distinct anion currents, ClC-3 and IClswell.