In this study, we describe the preparation and evaluation of new fluorescent sensor nanoparticles for the ratiometric measurement of chloride concentrations. Both a chloride-sensitive dye (lucigenin) and a reference dye (sulforhodamine derivative) were incorporated into polyacrylamide nanoparticles via inverse microemulsion polymerization and investigated for their response to chloride ions in buffered suspension as well as in living cells. The fluorescence intensity of lucigenin reversibly decreased in the presence of chloride ions due to a collisional quenching process, which can be described with the Stern-Volmer equation. The determined Stern-Volmer constant K SV for the quenching of lucigenin incorporated into particles was found to be 53 M (-1) and is considerably smaller than the Stern-Volmer constant for quenching of free lucigenin ( K SV = 250 M (-1)) under the same conditions. To test the nanosensors in living cells, we incorporated them into Chinese hamster ovary cells and mouse fibroblasts by using the conventional lipofectamin technique and monitored the response to changing chloride concentrations in the cell.