Abstract The protein-processing system of spinach leaf mitochondria was investigated using different mitochondrial fractions and in vitro transcribed and translated mitochondrial precursor proteins, the F 1β subunit of the F 0F 1-ATP synthases of Nicotiana plumbaginifolia and Neurospora crassa and the Neurospora Rieske FeS protein. Processing resulted in cleavage of the precursor proteins to the mature size products. The processing activity of spinach leaf mitochondria was found to be located in the mitochondrial membrane fraction. The activity could not be dissociated from the membrane by treatment of the membranes with 100 mM KCl, 4 M urea or at pH 11. The membrane-bound processing activity could not be stimulated by addition of the mitochondrial matrix fraction. These results show that the spinach leaf mitochondrial processing proteinase that catalyzes cleavage of the mitochondrial F 1β and Rieske FeS precursors is a membrane-associated protein. These results are in contrast to the results described for yeast, Neurospora and rat liver, where the corresponding processing proteinase was found to be a matrix enzyme. The membrane-bound processing activity of spinach leaf mitochondria was stimulated by the divalent cations Mn 2+, Zn 2+ and Co 2+ and inhibited by the metal chelators orthophenanthroline and EDTA, indicating that the processing proteinase is a metalloproteinase. Western blots of spinach leaf and potato tuber mitochondria incubated with antibodies against Neurospora processing enhancing protein (PEP) showed strong cross-reactivity with a protein of 63 kDa in spinach mitochondria and a protein of 59 kDa in potato mitochondria. Antibodies against Neurospora mitochondrial processing peptidase (MPP) did not cross-react with any spinach or potato mitochondrial proteins.