This study investigates the subcellular distribution of superoxide dismutase (SOD) in rat liver, focusing on the Cu,Zn-SOD and Mn-SOD activities. Rat liver homogenates were fractionated using differential centrifugation and equilibrium sedimentation in Nycomed gradients. The cytosol and lysosomal fractions contained Cu,Zn-SOD, while the mitochondrial matrix contained Mn-SOD. Surprisingly, Cu,Zn-SOD was found in the intermembrane space of mitochondria, and Mn-SOD was present in both the matrix and inner membrane. The Mn-SOD associated with the inner membrane was solubilized by 0.5 M NaCl, and the intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD that could be solubilized with detergents and NaCl. Both cytosolic and mitochondrial Cu,Zn-SODs were isolated and found to have identical properties, including molecular mass, cyanide sensitivity, SDS sensitivity, heat stability, and chloroform + ethanol stability. Tissue from Cu,Zn-SOD knockout mice was devoid of Cu,Zn-SOD, confirming that the cytosolic and intermembrane space Cu,Zn-SODs are coded by the same gene. The study discusses the significance of this distribution in protecting against superoxide damage.This study investigates the subcellular distribution of superoxide dismutase (SOD) in rat liver, focusing on the Cu,Zn-SOD and Mn-SOD activities. Rat liver homogenates were fractionated using differential centrifugation and equilibrium sedimentation in Nycomed gradients. The cytosol and lysosomal fractions contained Cu,Zn-SOD, while the mitochondrial matrix contained Mn-SOD. Surprisingly, Cu,Zn-SOD was found in the intermembrane space of mitochondria, and Mn-SOD was present in both the matrix and inner membrane. The Mn-SOD associated with the inner membrane was solubilized by 0.5 M NaCl, and the intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD that could be solubilized with detergents and NaCl. Both cytosolic and mitochondrial Cu,Zn-SODs were isolated and found to have identical properties, including molecular mass, cyanide sensitivity, SDS sensitivity, heat stability, and chloroform + ethanol stability. Tissue from Cu,Zn-SOD knockout mice was devoid of Cu,Zn-SOD, confirming that the cytosolic and intermembrane space Cu,Zn-SODs are coded by the same gene. The study discusses the significance of this distribution in protecting against superoxide damage.