Mitochondrial protein import is a complex multistep process from synthesis of proteins in the cytosol, recognition by receptorson the organelle surface, to translocation across one or both mitochondrial membranes and assembly after removal of thetargeting signal, referred to as a presequence. In plants, import has to further discriminate between mitochondria andchloroplasts. In this study,we determined the precise cleavage sites in the presequences for Arabidopsis (Arabidopsis thaliana) andrice (Oryza sativa) mitochondrial proteins using mass spectrometry by comparing the precursor sequences with experimentalevidence of the amino-terminal peptide from mature proteins. We validated this method by assessments of false-positive ratesand comparisons with previous available data using Edman degradation. In total, the cleavable presequences of 62 proteins fromArabidopsis and 52 proteins from rice mitochondria were determined. None of these proteins contained amino-terminalacetylation, in contrast to recent findings for chloroplast stromal proteins. Furthermore, the classical matrix glutamatedehydrogenase was detected with intact and amino-terminal acetylated sequences, indicating that it is imported intomitochondria without a cleavable targeting signal. Arabidopsis and rice mitochondrial presequences had similar isoelectricpoints, hydrophobicity, and the predicted ability to form an amphiphilic a-helix at the amino-terminal region of the presequence,but variations in length, amino acid composition, and cleavage motifs for mitochondrial processing peptidase were observed. Acombination of lower hydrophobicity and start point of the amino-terminal a-helix in mitochondrial presequences in bothArabidopsis and rice distinguished them (98%) from Arabidopsis chloroplast stroma transit peptides. Both Arabidopsis and ricemitochondrial cleavage sites could be grouped into three classes, with conserved 23R (class II) and 22R (class I) or without anyconserved (class III) arginines. Class II was dominant in both Arabidopsis and rice (55%–58%), but in rice sequences there wasmuch less frequently a phenylalanine (F) in the 21 position of the cleavage site than in Arabidopsis sequences. Our data alsosuggest a novel cleavage motif of (F/Y)Y(S/A) in plant class III sequences.