Herein we describe the development of the first mitochondrial Cl--selective fluorescent probe, Mito-MQAE, and its applications in biological systems. Fluorescence of Mito-MQAE is insensitive to pH over the physiological pH range and is quenched by Cl- with a Stern-Volmer quenching constant of 201 M-1 at pH 7.0. The results of cell studies using Mito-MQAE show that substances with the ability to disrupt mitochondrial membranes cause increases in the mitochondrial Cl- concentration.