Mitochondrial (mt) ribosomes and ribosomal subunits were prepared from HeLa S(,3) (chloramphenicol (CAP) sensitive) and 296-1 (CAP resistant) cell lines and used in in vitro protein synthesis activity studies. Mt ribosomes isolated in low salt (100 mM KCl, 30 mM MgCl(,2)) were active in poly(U)-directed protein synthesis when supplemented with E. coli tRNA and E. coli supernatant fraction (S-100). Washing these ribosomes with high salt-containing buffers (500 mM KCl, 5 mM MgCl(,2)) results in the loss of protein synthesis activity. Activity by high salt washed mt ribosomes was restored by the addition of a mt supernatant fraction (mt S-100);Mt S-100 was shown to aminoacylate both E. coli and mt tRNA with phenylalanine. The efficiency of this aminoacylation was not sufficient to support protein synthesis; E. coli S-100 was required in the protein synthesis system to provide sufficient aminoacyl-tRNA synthetase activity;Mt S-100 was found to inhibit protein synthesis by E. coli ribosomes. This inhibition was not due to contamination by lysosomes in mt preparations, nor was it affected by RNasin, a ribonuclease inhibitor. The cause of this inhibition remains unknown;Mt ribosomal subunits were prepared by dissociation in high salt and separation on sucrose gradients. Ribosomes, reconstituted from subunits, were active in polynucleotide-directed protein synthesis. Using hybrid ribosomes reconstituted from S(,3) and 296-1 subunits in poly(U,C)-directed protein synthesis, it was found that the cytoplasmicly inherited mutant gene conferring CAP resistance to the 296-1 cell line, alters a component of the mt ribosomal large subunit.