A study was made of factors thought to affect calcium retention by highly purified skeletal-muscle mitochondria. Substrate-supported (oxygen-dependent) and ATP-supported Ca2+ uptake were both examined. The effects of high concentrations of inorganic phosphate, inhibition of adenine nucleotide translocation and other influences on ATPase activity were also investigated. Results indicate that calcium is not released during anoxia if ATP is available to the mitochondrial ATPase. The data are discussed in relation to mitochondrial calcium transport in general, and its significance in post-mortem muscle.