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The Ca2+-indicator dye Arsenazo III has been injected into mouse oocytes by intracellular ionophoresis, and the optical absorbance of the injected dye measured using a double-beam microspectrophotometer. The absorbance spectrum shows a broad peak between 565 and 580 nm, with a maximum of 0.020-0.095 in different oocytes. From these values, intracellular dye concentrations were estimated to be 0.1-0.5 mM, although estimated concentrations up to 1.0 mM have been used in later experiments where the dye absorbance was measured only at selected wave-lengths. The transport number for intracellular ionophoresis of the dye has been estimated. The mean value was 0.009 in experiments where the injection current was allowed to cross the cell membrane, but was only 0.0015 when the injection current was balanced by a current of opposite polarity through a second electrode barrel. In oocytes exposed to the mitochondrial uncoupler DNP (2,4-dinitrophenol), the shape of the Arsenazo III absorbance spectrum was found to change. The resulting difference spectra show the peaks at about 610 and 660 nm which are characteristic features of the Arsenazo III Ca2+-difference spectrum. These absorbance changes were not completely reversible on removal of DNP. Changes in the optical absorbance of Arsenazo III have been observed in oocytes injected with Ca2+ by intracellular ionophoresis. Estimated increases of intracellular free [Ca2+], which were of the order of 1 microM and decayed within 30 s, are discussed in relation to the Ca2+-buffering and Ca2+-elimination properties of the oocyte.