Previously, the double-transposon (Tn) mutant VAN20 of Vibrio anguillarum (Va) 775.17B was isolated. This mutant lacked a major surface antigen (MSA) suggested to be a lipopolysaccharide (LPS) and showed a 10(5)-fold increase in the 50% lethal dose (LD50) when fish were infected intraperitoneally. In this study, the two Tn insertion sites within the chromosome were identified, a plasmid insertion mutation was made at each locus in a more virulent strain of Va, NB10, and the virulence was analyzed. One mutant displayed a 10(4)-fold increase in LD50, whereas the second mutant showed the wild-type (wt) phenotype. However, both mutants still expressed the MSA, suggesting that there may be more than two Tn insertions in VAN20 or that a double mutation is required to prevent production of the MSA. The DNA locus for the virulent phenotype was cloned and sequenced. A potential transcriptional unit consisting of three putative open reading frames (ORFs) was identified. The Tn was located in the second ORF, virC (virulence). The first ORF (34.8 kDa) showed 30% homology to the Escherichia coli and Salmonella typhimurium cysG (cysteine) genes. The virC gene (51.4 kDa) and the third ORF (24 kDa) showed no homology to other proteins in GenBank. Plasmid insertion mutants were made within each of these ORFs and the virulence was assayed. Only the virC mutant showed a loss in virulence, indicating that virC is a novel gene that is essential for the virulence of Va.