Cardiolipin, a lipid of the mitochondrial inner membrane, is lost from many types of cells during apoptotic death. Here we show that the cardiolipin content of nerve growth factor (NGF)-deprived rat sympathetic neurons undergoing apoptotic death in cell culture decreased before extensive loss of mitochondria from the cells. By 18-24 h after NGF deprivation, many neurons did not stain with the cardiolipin-specific dye, Nonyl Acridine Orange, suggesting complete loss of cardiolipin. Gas chromatography confirmed the decline of cardiolipin content in NGF-deprived neurons. Electron microscopy and immunoblots for the mitochondrial-specific protein, heat shock protein 60 (HSP60), revealed that there was only a slight decrease in mitochondrial mass at this time. Cardiolipin loss after NGF deprivation was concurrent with increased production of mitochondrial-derived reactive oxygen species [Kirkland, R.A., Franklin, J.L., 2001. J. Neurosci. 21, 1949-1963] and increased lipid peroxidation. Compounds having antioxidant effects blocked peroxidation, loss of cardiolipin, and the decrease of mitochondrial mass in NGF-deprived neurons. These compounds also blocked an increase in the number of lysosomes and autophagosomes in NGF-deprived cells. The findings reported here show that the important mitochondrial inner membrane lipid, cardiolipin, is lost from mitochondria during neuronal apoptosis and that this loss occurs before significant loss of mitochondria from cells. They suggest that the loss of cardiolipin is mediated by free radical oxygen.