Mitochondrial proteins soluble in neutral chloroform -methanol (2 : 1) were separated from lipids by ether precipitation and resolved by Sephadex G-200 filtration in the presence of dodecylsulfate into two major fractions eluting in the excluded region (peak I) and in a region of an apparent molecular weight 8000 (peak 11). Residual phospholipids are found only in peak 11. Peak I consists of several aggregated small polypeptides of molecular weights around 8000, which can be disaggregated by mild oxidation with performic acid.Cycloheximide stimulates almost two-fold incorporation of radioactive phenylalanine into peak I proteins but inhibits labelling of peak I1 proteins by 95 %. Chloramphenicol and ethidium bromide inhibit the synthesis of peak I proteins by 70 % and 95 % respectively, but do not affect labelling of peak I1 proteins.At least 30 % of the translation products of mitochondrial DNA in vitro behave like peak I proteins :they are soluble in organic solvents, they aggregate in dodecylsulfate buffer after removal of phospholipids and they contain species of molecular weights around 8000 that disaggregate upon oxidation. The data strongly suggest that the proteins of peak I are encoded by mitochondrial genes and synthesized on mitochondrial ribosomes, whereas the proteins of peak I1 are encoded by nuclear genes and synthesized on cytoplasmic ribosomes.Both groups of lipophilic proteins are very similar in their molecular weights, but the mitochondrially coded peak I proteins have the unique property of forming large heat-stable aggregates in the presence of dodecylsulfate.Proteins soluble in organic solvents are generally believed to penetrate into the lipid bilayer of biomembranes and are therefore called "integral" or "intrinsic" membrane proteins [l, 21. Most proteins synthesized within mitochondria belong to this class because they are soluble either in acid or in neutral chloroform -methanol (2 : 1) [3 -81. Those mitochondrial translation products that are soluble in neutral chloroform -methanol have rather low molecular weights of around 8000 [3,6,8] and include a component of the "membrane fraction" of oligomycinsensitive ATPase in yeast [9]. A protein fraction of similar size and solubility properties has been synthesized in vitro in cell-free systems programmed with mitochondrial DNA or mitochondrial messenger RNA from Neurospora crassa [lo].This suggests that at least some mitochondrial genes code for small hydrophobic proteins, which penetrate the lipid phase of the inner mitochondrial membrane. However, this group of proteins could be quite heterogeneous and it is still open as to how many species are contributed by the mitochondrial or by the nuclear : cytoplasmic genetic system. In order to answer these questions and to study structure and function of individual intrinsic membrane proteins we have started to isolate and purify from Neurospora crassa mitochondrial proteins soluble in neutral chloroform -methanol (2 : 1).It turned out that most of these proteins are in a rather narrow molec...