Most mammalian cell types, including endothelial cells, respond to cell swelling by activating a Cl − current termed I Cl,swell , but it is not known how the physical stimulus of cell swelling is transferred to the channels underlying I Cl,swell . We have investigated the precise relation between cell volume and I Cl,swell in endothelial cells by performing whole-cell current recordings while continuously monitoring cell thickness ( T c ) as a measure for cell volume. The time course of T c was accurately predicted by a theoretical model that describes volume changes of patch-clamped cells in response to changes in the extracellular osmolality (OSM o ). This model also predicts significant changes in intracellular ionic strength (Γ i ) when OSM o is altered. Under all experimental conditions I Cl,swell closely followed the changes in Γ i , whereas I Cl,swell and cell volume were often found to change independently. These results do not support the hypothesis that Γ i regulates the volume set point for activation of I Cl,swell . Instead, they are in complete agreement with a model in which a decrease of Γ i rather than an increase in cell volume is the initial trigger for activation of I Cl,swell .