The elaboration, distribution, and fate of newly formed proteins in mitochondria were studied by quantitative radioautography combined with electron microscopy. Young and adult rats were sacrificed at intervals between 2 minutes and 6 hours after intravenous or intraperitoneal injection of l-leucine-3H or dl-lysine-3H. Time intervals of 8, 12, and 16 days were used in another experiment after repetitive injections of dl-lysine-3H and dl-phenylalanine-3H. In the radioautographs, the number of silver grains was determined per unit area (radioactivity concentration) for the different organelles of parenchymal cells of the liver and proximal convoluted tubule cells of the kidney. As early as 2 min after injection, mitochondria were found to be labeled, irrespective of their cytoplasmic localization. The radioactivity concentration increased rapidly within the 10 min after the injection. Decay curves of radioactivity examined between 8 and 16 days provide an average half-life of 9.4 days for the mitochondrial proteins. The distribution of the silver grains counted over the mitochondrial sections was statistically analyzed and found to be randomized: these results provide a strong argument in favor of a local synthesis and a continual turnover of protein in mitochondria. It is suggested that the mitochondrial components may be renewed by replacement of subunits. © 1969 Academic Press, Inc.