Roots are highly sensitive organs in plants. To gain a better knowledge of the chilling stress responses of plants, it is imperative to analyze the tissue-specific proteome patterns under chilling stress. In the present study, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry, has been adopted to investigate the protein expression patterns of rice roots in response to chilling stress. Rice seedlings were subjected to 10 degrees C and samples were collected 24 and 72h after treatment. To identify the low-abundant proteins in root tissues, samples were fractionated by 15% polyethylene glycol (PEG), separated by 2-DE, and visualized by silver or CBB staining. A total of 27 up-regulated proteins were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry or electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analysis. Together with the previously identified cold-stress-responsive proteins, a group of novel proteins were identified including acetyl transferase, phosphogluconate dehydrogenase, NADP-specific isocitrate dehydrogenase, fructokinase, PrMC3, putative alpha-soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, and glyoxalase 1. These proteins are involved in several cellular processes, including energy production and metabolism, vesicular trafficking, and detoxification. Gene expression at the mRNA level of some selected proteins revealed that transcription levels are not always concomitant to the translational level. Thus, investigation of root proteome expression and identification of some novel proteins could be useful in better understanding the molecular basis of chilling stress responses in plants.