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.