The virulence region of the Salmonella enteritidis 55 kb plasmid pNL2001 was identified by Tn1-insertion mutagenesis, DNA hybridization studies, and Western blot analysis of proteins encoded in the virulence region of the plasmid. DNA hybridization studies showed that the pNL2001 plasmid contained a 6.4 kb SalI-EcoRI fragment homologous to the 6.4 kb SalI-EcoRI Salmonella plasmid virulence (spv) region of the S. choleraesuis 50 kb plasmid (pKDSC50). One of the 247 Tn1-insertion mutants of S. enteritidis, designated strain TA19, showed a reduced mouse lethality, and the Tn1-insertion of strain TA19 was located within this homologous 6.4 kb region, suggesting that the 6.4 kb SalI-EcoRI fragment of pNL2001 contained the spv region. Two contiguous SalI-EcoRI fragment were cloned into the expression vectors. By the 6.4 kb SalI-EcoRI fragment were cloned into the expression vectors. By Western blot analysis using four Spv peptide antisera, each specific for individual proteins encoded in the spvR, spvA, spvB and spvC genes of pKDSC50, four proteins encoded in the 6.4 kb SalI-EcoRI fragment of pNL2001 were identified. Protein SpvR with an apparent molecular mass of 32 kDa was produced from the 2.3 kb SalI-EcoRI fragment, and protein SpvA, SpvB and SpvC with apparent molecular masses of 32, 70 and 29 kDa, respectively, were produced from the 4.1 kb EcoRI-EcoRI fragment. From the 4.1 kb EcoRI::Tn1 fragment of the TA19 plasmid, proteins SpvA and SpvB were expressed, but not SpvC. It was therefore suggested that the spvC gene may contribute to the expression of virulence of S. enteritidis. Furthermore, the nucleotide sequence of the 6.4 kb SalI-EcoRI fragment encoding these four proteins was determined. Four open reading frames which encoded the four proteins with deduced molecular masses of 33,906, 28,200, 65,349 and 27,646 Da were detected. Deduced amino acid sequences of each protein showed a high degree of identity to corresponding sequences in the virulence region of S. choleraesuis, S. dublin and S. typhimurium virulence plasmids. Therefore, we confirmed that the virulence plasmids of Salmonellae including S. enteritidis share the highly conserved region responsible for virulence.