Mitochondria can synthesize a limited number of proteins encoded by mtDNA (mitochondrial DNA) by using their own biosynthetic machinery, whereas most of the proteins in mitochondria are imported from the cytosol. It could be hypothesized that the mitochondrial pool of amino acids follows the frequency of amino acids in mtDNA-encoded proteins or, alternatively, that the profile is the result of the participation of amino acids in pathways other than protein synthesis (e.g. haem biosynthesis and aminotransferase reactions). These hypotheses were tested by evaluating the pool of free amino acids and derivatives in highly-coupled purified liver mitochondria obtained from rats fed on a nutritionally adequate diet for growth. Our results indicated that the pool mainly reflects the amino acid composition of mtDNA-encoded proteins, suggesting that there is a post-translational control of protein synthesis. This conclusion was supported by the following findings: (i) correlation between the concentration of free amino acids in the matrix and the frequency of abundance of amino acids in mtDNA-encoded proteins; (ii) the similar ratios of essential-to-non-essential amino acids in mtDNA-encoded proteins and the mitochondrial pool of amino acids; and (iii), lack of a correlation between codon usage or tRNA levels and amino-acid concentrations. Quantitative information on the mammalian mitochondrial content of amino acids, such as that presented in the present study, along with functional studies, will help us to better understand the pathogenesis of mitochondrial diseases or the biochemical implications in mitochondrial metabolism.