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