Prior investigation of the protein synthesizing properties of mitochondria involved the whole organelle. In order to better characterize these properties, the present study was concerned more specifically with the activity of the inner mitochondrial membranes (IMM) which recent investigation has implicated as the primary location of mitochondrial ribosomes. To further define mitochondrial protein synthesis simultaneous experimentation was also conducted utilizing cytoplasmic ribosomes thus enabling both qualitative and quantitative comparison between the two systems. Results from this series of investigations reveal a dramatic amino acid incorporating ability by the IMM fraction of the brain mitochondria. This activity, in turn, was shown to be highly independent of exogenous sources of ATP, GTP, pH 5 enzymes, and cytoplasmic ribosomes. Furthermore, the addition of an exogenous source of messenger RNA, polyuridylic acid or (poly (U)) which resulted in an increased incorporation of [14C]phenylalanine into polypeptide in the cytoplasmic system was found to have no effect on the IMM system. Upon comparison of the in vitro protein synthesizing properties of the IMM fraction with those of the cytoplasmic ribosomal system, it became evident that obvious differences existed in the degree of amino acid incorporation and in the sensitivity of this process to the various protein synthesizing inhibitors. Cytoplasmic ribosomes demonstrated a much greater [14C]leucine and [14C]phenylalanine incorporating activity than the IMM fraction. In addition, RNase and cyclohexamide had their greatest effect on the cytoplasmic system while the action of chloramphenicol was most potent on the IMM system. Although puromycin inhibited both protein synthesizing systems, this effect was greatest in the presence of cytoplasmic ribosomes.