Nuclear proteins play key roles in the fundamental regulation of genome instability, the phases of organ development, and physiological responsiveness through gene expression. Although nuclear proteins have been shown to account for approximately one-fourth of total proteins in yeast, no efficient method to identify novel nuclear proteins has been applied to plants. In this study, a trial to isolate nuclear proteins in rice was attempted, and several novel nuclear proteins showing a variety of subnuclear localizations were identified. The nuclear transportation trap (NTT) system, which is a modified two-hybrid system, isolated many nuclear proteins from rice (Oryza sativa) NTT cDNA libraries. Nuclear localization of the isolated proteins was confirmed by transient introduction of green fluorescent protein fusion constructs for a subset of protein genes into onion (Allium cepa) cells. The majority of these proteins, including novel proteins and proteins initially categorized as cytoplasmic proteins, were revealed to be localized in the nucleus. Detailed characterization of unknown proteins revealed various subnuclear localizations, indicating their possible association with chromatin and the nuclear matrix with a foci or speckle-like distribution. Some also showed dual distribution in the nucleus and cytoplasm. In the novel protein fraction, a protein was further identified for its chromatin-associated localization in a specific organ of rice by immunostaining. Thus, a variety of novel nuclear architectural proteins with chromatin or matrix associating abilities, which are important in nuclear organization by influencing certain organ developments or cell responsiveness, can be isolated using the NTT method. Because nuclear proteins other than transcription regulators have rarely been characterized in plants, such as matrix proteins and development-specific chromatin proteins, their identification and subsequent characterization could provide important information for genome-wide regulatory mechanisms controlled by nuclear organization.