This paper reports the characterization of a new locus, vagC/vagD, on the virulence plasmid of Salmonella dublin. Strain G19, harbouring a TnA insertion in vagC, exhibited reduced virulence although vagC was outside the 8 kb essential virulence region. G19 was also unable to grow on minimal-medium containing various sole carbon/energy sources, unlike the wild-type and plasmid-cured strains. Sequencing of the locus revealed the presence of two ORFs (vagC and vagD) which overlapped by one nucleotide. The VagC polypeptide (12 kDa) was observed using minicell expression. Results indicated that vagD was responsible for the phenotypic differences observed between the wild type and G19, and that vagC modulated the activity of vagD. Furthermore, microscopic analysis of G19 cells harvested from minimal-medium plates showed that a high proportion of cells were elongated, which suggested that vagC and vagD might be involved in coordination of plasmid replication with cell division. We propose that vagD, under certain environmental conditions, acts to prevent cell division until plasmid replication is complete, thus aiding plasmid maintenance. vagC and vagD are absent from the related virulence plasmid of Salmonella typhimurium.