Targeting of DNA molecules to specific subcellular positions is essential for efficient segregation, but the mechanisms underlying these processes are poorly understood. In Escherichia coli, several plasmids belonging to different incompatibility groups (F, P1 and RK2) localize preferentially near the midcell and quartercell positions. Here we compare the relative positions of these three plasmids using fluorescence in situ hybridization. When plasmids F and P1 were localized simultaneously using differentially labeled probes, the majority of foci (approximately 75%) were well separated from each other. Similar results were found when we compared the subcellular localization of F with RK2, and RK2 with P1: regardless of the number of foci per cell or growth conditions, most of the foci (70-80%) were not in close proximity to one another. We also localized RK2 in Pseudomonas aeruginosa and Vibrio cholerae, and found that plasmid RK2 localization is conserved across bacterial species. Our results suggest that each plasmid has its own unique subcellular address, implying a mechanism for the stable co-existence of plasmids in which subcellular targeting plays a major role.