Purified rice (Oryza sativa) mitochondrial proteins have been arrayed by isoelectric focusing/polyacrylamide gel electrophoresis (PAGE), by blue-native (BN) PAGE, and by reverse-phase high-performance liquid chromatography (LC) separation (LC-mass spectrometry [MS]). From these protein arrays, we have identified a range of rice mitochondrial proteins, including hydrophilic/hydrophobic proteins (grand average of hydropathicity = -1.27 to +0.84), highly basic and acid proteins (isoelectric point = 4.0-12.5), and proteins over a large molecular mass range (6.7-252 kD), using proteomic approaches. BN PAGE provided a detailed picture of electron transport chain protein complexes. A total of 232 protein spots from isoelectric focusing/PAGE and BN PAGE separations were excised, trypsin digested, and analyzed by tandem MS (MS/MS). Using this dataset, 149 of the protein spots (the products of 91 nonredundant genes) were identified by searching translated rice open reading frames from genomic sequence and six-frame translated rice expressed sequence tags. Sequence comparison allowed us to assign functions to a subset of 85 proteins, including many of the major function categories expected for this organelle. A further six spots were matched to rice sequences for which no specific function has yet been determined. Complete digestion of mitochondrial proteins with trypsin yielded a peptide mixture that was analyzed directly by reverse-phase LC via organic solvent elution from a C-18 column (LC-MS). These data yielded 170 MS/MS spectra that matched 72 sequence entries from open reading frame and expressed sequence tag databases. Forty-five of these were obtained using LC-MS alone, whereas 28 proteins were identified by both LC-MS and gel-based separations. In total, 136 nonredundant rice proteins were identified, including a new set of 23 proteins of unknown function located in plant mitochondria. We also report the first direct identification, to our knowledge, of PPR (pentatricopeptide repeat) proteins in the plant mitochondrial proteome. This dataset provides the first extensive picture, to our knowledge, of mitochondrial functions in a model monocot plant.