Gel electrophoretic analysis of mitochondrial membranes from Neurospora crassa shows the presence of a polypeptide fraction with apparent molecular weights of 7000 - 1200, which is synthesized on mitochondrial ribosomes. This fraction comprises between 10 and 50% of total mitochondrial translation products. Evidence is presented that the major part of this fraction is derived from components with higher apparent molecular weights by proteolytic activity. The proteolytic activity is located in vesicles which are co-isolated with mitochondria upon differential centrifugation. The activity is strongly enhanced by application of detergents such as sodium dodecylsulfate and Triton. Proteins synthesized on mitochondrial as well as cytoplasmic ribosomes are subject to proteolytic breakdown. This proteolysis can be blocked by addition of inhibitors such as diisopropylfluorphosphate to isolated mitochondria. Similar observations were made with Schizosaccharomyces pombe. In Neurospora, the amount of mitochondrial translation products with apparent molecular weights of less than 12000 is low in mitochondria from cells treated with cycloheximide for 1 h and high in mitochondria from cells treated with cycloheximide for 5 min. This observation is explained by the finding that proteinase activity in mitochondrial preparations decreases exponentially with a t1/2 of 20 min during preincubation of cells with cycloheximide. Procedures are described to remove or block contaminating proteinase activity. The results appear to be relevant for the interpretation of many data obtained from experiments in which this puzzling kind of artifact has not been sufficiently considered.