Bacteria-free mitochondria were isolated from aseptically grown, etiolated and green seedlings of both cytoplasmic male-sterile (WA-type) and male-fertile rice (Oryza sativa L.). Protein synthesis in these isolated mitochondria was characterized by gel electrophoresis/fluorography and by the incorporation of [(35)S]-methionine into protein. In the presence of cycloheximide, a set of some 25 discrete polypeptides and an electrophoretically unresolved population were synthesized. This pattern of protein synthesis in organello was essentially the same in mitochondria isolated from both male-fertile and malesterile cytoplasms. Our data does not preclude the possibility, however, that the WA-type CMS possesses a tissue-specific and/or a low abundance mitochondrial protein(s), whose synthesis eluded detection under our experimental conditions. The synthesis of the mitochondria-encoded polypeptides by isolated rice mitochondria was inhibited by chloramphenicol and incompletely inhibited by erythromycin. A minor chloramphenicol-insensitive, cycloheximide-sensitive translation activity was found consistently to copurify with the mitochondria. This activity generated a reproducible electrophoretic profile of a poorly resolved, weakly labelled population of polypeptides and of a few conspicuous polypeptides, including a 42 kDa species.