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A novel long wavelength fluorescent Cl⁻ indicator was used to test whether endosomal Cl⁻ conductance provides the principal electrical shunt to permit endosomal acidification. The green fluorescent Cl⁻-sensitive chromophore 10,10′-bis[3-carboxypropyl]-9,9′-biacridinium dinitrate (BAC) was conjugated to aminodextran together with the red fluorescent Cl⁻-insensitive chromophore tetramethylrhodamine (TMR). BAC fluorescence is pH-insensitive and quenched by Cl⁻with a Stern-Volmer constant of 36 m⁻¹. Endosomes in J774 and Chinese hamster ovary (CHO) cells were pulse-labeled with BAC-TMR-dextran by fluid-phase endocytosis. Endosomal [Cl⁻] increased over 45 min from 17 to 53 mm in J774 cells and from 28 to 73 mm in CHO cells, during which time endosomal pH decreased from 6.95 to 5.30 (J774) and 6.92 to 5.60 (CHO). The acidification and increased [Cl⁻] were blocked by bafilomycin …