Tetradecameric Clp protease core complexes in nonphotosynthetic plastids of roots, flower petals, and in chloroplasts of leaves of Arabidopsis thaliana were purified based on native mass and isoelectric point and identified by mass spectrometry. The stoichiometry between the subunits was determined. The protease complex consisted of one to three copies of five different serine-type protease Clp proteins (ClpP1,3-6) and four non-proteolytic ClpR proteins (ClpR1-4). Three-dimensional homology modeling showed that the ClpP/R proteins fit well together in a tetradecameric complex and also indicated unique contributions for each protein.Lateral exit gates for proteolysis products are proposed. In addition, ClpS1,2, unique to land plants, tightly interacted with this core complex, with one copy of each per complex. The three-dimensional modeling show that they do fit well on the axial sites of the ClpPR cores. In contrast to plastids, plant mitochondria contained a single ϳ320-kDa homo-tetradecameric ClpP2 complex, without association of ClpR or ClpS proteins. It is surprising that the Clp core composition appears identical in all three plastid types, despite the remarkable differences in plastid proteome composition. This suggests that regulation of plastid proteolysis by the Clp machinery is not through differential regulation of ClpP/R/S gene expression, but rather through substrate recognition mechanisms and regulated interaction of chaperone-like molecules (ClpS1,2 and others) to the ClpP/R core.Plastids are essential organelles of prokaryotic origin that are present in every plant cell and differentiate from proplastids into non-photosynthetic plastids in roots and flowers and photosynthetic plastids in leafs and stems. Plastids are responsible for synthesis of key molecules required for the architecture and functions of plant cells.To maintain a correct stoichiometry between different proteins and pathways, to remove and recycle damaged or misfolded proteins, and to control gene expression by proteolysis of transcription or translation factors, different proteolytic systems are present in the plastid. Members of at least five protease families are present in plastids, but their structures, functions, substrates, and biological importance are poorly understood (2).A very prominent group of proteases in plants is the Clp protease family. Our latest analysis of the Arabidopsis thaliana nuclear genome indicates the presence of at least 26 Clp-related genes, with 15 genes encoding for plastid-localized proteins (3) (Fig.