This protocol describes the purification of mitochondria from rat liver with the aid of zone electrophoresis in a free flow device (ZE-FFE). Starting from liver homogenate, cell debris and nuclei are removed by low speed centrifugation. A crude mitochondrial fraction is obtained by medium speed centrifugation and is further purified by washing followed by a Nycodenz^® gradient centrifugation. Lysosomes and microsomes are located at the upper parts of the gradient, whereas mitochondria are found in the medium part of the gradient. A subsequent purification step with ZE-FFE efficiently removes remaining lysosomes and microsomes and, importantly, damaged mitochondrial structures. The resulting purified mitochondria can be concentrated by centrifugation and used for further experiments. Finally, possible modifications of this protocol with respect to the isolation of pure lysosomes are discussed.