Ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I) is an important initial step for the production of ribosomes. The RNA polymerase 1-2 (Rpo1-2) gene is comprised of 15 exons and encodes 1135 amino acids (aa) of the second largest subunit in Pol I. In a gene trap screen, we have identified an insertional mutation (Rpo1-2(Gt)) in the 14th exon of Rpo1-2, resulting in a truncation of 312aa from the C-terminal. In Rpo1-2(Gt/Gt) embryos, the synthesis of rRNA was severely impaired. Rpo1-2(Gt/Gt) embryos could develop to the morula stage, and thereafter displayed nucleolus disruption and apoptotic cell death. These results indicate that the loss of rDNA transcription induced nucleolar structure disorganization and apoptosis in preimplantation embryos.