The mitochondrial processing peptidase (MPP) of Neurospora crassa consists of two subunits termed alpha-and beta-MPP. Here we present spectroscopic and chromatographic studies indicative of adenine nucleotide binding in the two MPP subunits. ADP was identified as the cofactor of alpha-MPP and ATP as the cofactor of beta-MPP. The nucleotides are not covalently bound and exert strong control on the conformational and functional properties of the subunits. The ADP cofactor of alpha-MPP seems to be of utmost importance for the proteolytic activity because no processing of the precursor protein was registered in the assay containing alpha-MPP depleted of ADP and native beta-MPP. On the contrary, with native alpha-MPP and depleted of ATP beta-MPP almost 100% processing activity could be measured. Very strong increase of the intensity and significant changes in the shape and maximum position of the protein fluorescence spectra were observed after removal of the adenine cofactors of alpha- and of beta-MPP.