The origin of eukaryotic cell nuclei by symbiosis of Archaea in Bacteria was proposed on the basis of the phylogenetic topologies of genes. However, it was not possible to conclude whether or not the genes involved were authentic representative genes. Furthermore, using the BLAST and FASTA programs, the similarity of open reading frame (ORF) groups between three domains (Eukarya, Archaea and Bacteria) was estimated at one threshold. Therefore, their similarities at other thresholds could not be clarified. Here we use our newly developed 'homology-hit analysis' method, which uses multiple thresholds, to determine the origin of the nucleus. We removed mitochondria-related ORFs from yeast ORFs, and determined the number of yeast orthologous ORFs in each functional category to the ORFs in six Archaea and nine Bacteria at several thresholds (E-values) using the BLAST. Our results indicate that yeast ORFs related to the nucleus may share their origins with archaeal ORFs, whereas ORFs that are related to the cytoplasm may share their origins with bacterial ORFs. Our results thus strongly support the idea of nucleus symbiosis.