Nop25 is a putative RNA binding nucleolar protein associated with rRNA transcription. The present study was undertaken to determine the crucial function of Nop25 in the nucleolus. When N-terminal amino acids of Nop25 were overexpressed as a dominant negative effector in cells, the nucleolus was fragmented into small components. Knockdown of Nop25 by RNA interference also induced drastic nucleolar fragmentation. These results suggest that Nop25 is involved in nucleolar architecture and maintenance. Although nucleolar fragmentation induced the dispersion of nucleolar proteins from the nucleolus, it did not lead to cell cycle arrest or apoptosis, suggesting no effect of nucleolar fragmentation on the transcription and processing of rRNA molecules and subsequent ribosome biogenesis. Studies on Nop25 may provide new information on nucleolar organization related to nucleolar architecture and function. (C) 2007 Elsevier Inc. All rights reserved.