Mitochondrial preparations derived from denervated rat skeletal muscle and paired controls were characterized with respect to their ability to take up externally added Ca2+. The denervated and control muscle homogenates and mitochondrial [Ca2+] were also determined. Our data indicate that the denervated mitochondria are able to take up less Ca2+ than the controls before uncoupling occurs. This defect is associated with elevated [Ca2+] in homogenate and mitochondrial fractions in the denervated state. The causal relationship between Ca2+ overload, mitochondrial functional damage and cell necrosis is discussed.