The effects of various antibiotics on protein synthesis systems in procyclic trypomastigotes of Trypanosoma brucei brucei LUMP 1026 have been determined in an attempt to identify a mitochondrial translation system. The function of cytoplasmic ribosomes in T. b. brucei in vivo is sensitive to inhibition not only by cycloheximide but also by high concentrations of D-chloramphenicol, L-chloramphenicol, erythromycin and tetracycline as a possible secondary consequence of a primary inhibition of mitochondrial respiration in vivo by these latter four compounds. In support of this conclusion, antimycin A inhibits mitochondrial respiration and, secondarily, cytoplasmic protein synthesis in vivo, suggesting that mitochondrial phosphorylation is necessary for cytoplasmic translation in T. b. brucei. Tetracycline inhibition of cytoplasmic protein synthesis in vivo may also be due to a direct effect on cytoplasmic ribosomes, since this drug inhibits ribosome function in vivo. Low concentrations of D-chloramphenicol, erythromycin and tetracycline do not inhibit cycloheximide-insensitive protein synthesis in vivo, implying the presence of permeability barriers to these drugs in T. b. brucei. Mitochondrial fractions isolated from T. b. brucei possess a ribosomal translation system, the function of which is insensitive to cycloheximide but sensitive to inhibition by low concentrations of D-chloramphenicol should only be used with caution in studies of mitochondrial biogenesis in T. b. brucei.