HeLa cell mitochondrial polysomes, estimated to consist of two to seven 60S monomers, exhibit an unusual resistance to RNase and EDTA, possibly related to the strongly hydrophobic nature of their polypeptide products which makes the nascent chains particularly "sticky". Mitochondria-specific ribosomal and 4S RNA are coded for by mitochondrial DNA (mit-DNA): there is one gene for each ribosomal RNA (rRNA) species and nine genes for 4S RNA on the heavy (H) mit-DNA strand, and three 4S RNA genes on the light (L) strand. The existence of messenger RNA (mRNA) transcribed from mit-DNA is strongly suggested by the occurrence in mitochondria of poly(A) stretches, 60 to 80 residues long, most of which are covalently linked to RNA molecules coded for by mit-DNA. Mitochondrial poly(A) is not a transcription product of mit-DNA. The in vivo and in vitro products of mitochondrial protein synthesis exhibit by polyacrylamide gel electrophoresis a group of not well resolved components in the molecular weight range from 12 000 to 25 000 daltons, and another group, more abundant, in the range from 40 000 to 55 000 daltons. A strong constraint appears to exist in the pattern of amino acid utilization by HeLa cell mitochondria, with a very low level, if any, of incorporation of the charged polar amino acids, and a predominant utilization of the most hydrophobic ones. Block of mitochondrial protein synthesis by chloramphenicol for up to four generations of cell growth has no effect on mitochondrial growth and replication, on the rate of mitochondrial nucleic acid synthesis, and on the formation of a functional protein synthesizing apparatus; however, mitochondrial protein synthesis is required for the formation of a functional cytochrome c oxidase. No significant differences in the rate of mitochondrial protein synthesis per unit of cell mass, nor in the size distribution of the protein products, were found in HeLa cells in different phases of the cell cycle.