The steady-state unsaturated fatty acid content of yeast mitochondria was controlled by growing cells of an unsaturated fatty acid auxotroph under derepressed conditions in glucose-limited chemostat cultures. Variations in mitochondrial unsaturated fatty acid content influenced the activity and assembly of the mitochondrial protein-synthesizing system. The temperature of discontinuity of Arrhenius plots of in vitro mitochondrial protein synthetic activity increased from below 6 ° to 24 °C as the proportion of membrane unsaturated fatty acid was lowered from 75 to 44%. Arrhenius plots of the inner mitochondrial membrane functions ATPase and NADH oxidase gave similar results. When the proportion of membrane unsaturated fatty acid was reduced below about 30%, no in vitro mitochondrial protein synthetic activity could be detected. These mitochondria lacked cytochrome aa3, had low levels of cytochrome b, and the ATPase was insensitive to oligomycin inhibition, indicating that mitochondrial protein synthesis was also inactive in vivo. No mitochondrial ribosomal subunits could be detected in mitochondria containing 28% or less of their fatty acids unsaturated. Although mitochondrial transcription occurred, the RNA fractions isolated were all of low molecular weight, and intact mitochondrial ribosomal RNA could not be detected.