AbstractThe eukaryotic nucleus has been proposed to be organized by two interdependent nucleoprotein structures, the DNA‐based chromatin and the RNA‐dependent nuclear matrix. The functional composition and molecular organization of the second component have not yet been resolved. Here, we describe the isolation of the nuclear matrix from the model plant Arabidopsis, its initial characterization by confocal and electron microscopy, and the identification of 36 proteins by mass spectrometry. Electron microscopy of resinless samples confirmed a structure very similar to that described for the animal nuclear matrix. Two‐dimensional gel electrophoresis resolved approximately 300 protein spots. Proteins were identified in batches by ESI tandem mass spectrometry after resolution by 1D SDS–PAGE. Among the identified proteins were a number of demonstrated or predicted Arabidopsis homologs of nucleolar proteins such as IMP4, Nop56, Nop58, fibrillarins, nucleolin, as well as ribosomal components and a putative histone deacetylase. Others included homologs of eEF‐1, HSP/HSC70, and DnaJ, which have also been identified in the nucleolus or nuclear matrix of human cells, as well as a number of novel proteins with unknown function. This study is the first proteomic approach towards the characterization of a higher plant nuclear matrix. It demonstrates the striking similarities both in structure and protein composition of the operationally defined nuclear matrix across kingdoms whose unicellular ancestors have separated more than one billion years ago. J. Cell. Biochem. 90: 361–378, 2003. © 2003 Wiley‐Liss, Inc.