The nucleus is the subcellular organelle that contains nearly all the genetic information required for the regulated expression of cellular proteins. In this study, we comprehensively characterized the Arabidopsis nuclear proteome. Nuclear proteins were isolated and analyzed using two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Approximately 500-700 spots were detected in reference 2D gels of nuclear proteins. Proteomic analyses led to the identification of 184 spots corresponding to 158 different proteins implicated in a variety of cellular functions. We additionally analyzed the changes in the nuclear proteome in response to cold stress. Of the 184 identified proteins, 54 were up- or downregulated with a greater than twofold change in response to cold treatment. Among these, six proteins were selected for further characterization. Northern analysis data revealed that gene expression of these proteins was also altered by cold stress. Following transient expression in BY-2 protoplasts, two proteins were detected in both the cytoplasm and the nucleus and four others were detected exclusively in the nucleus, which correlates well with the nuclear localization patterns of the proteomic data. Our study provides an initial insight into the Arabidopsis nuclear proteome and its response to cold stress.