A fractionation procedure has been developed to separate mitochondrial membrane components into well-resolved fractions. One fraction of the membrane, which can be isolated by gel electrophoresis, accounts for a major portion of the total mass of the membrane protein. This protein was labeled to a much greater extent than other fractions during protein synthesis by isolated mitochondria. Inhibition of the synthesis of this component by chloramphenicol accounts for essentially all of the observed effect of chloramphenicol on protein biosynthesis in isolated mitochondria. © 1969.