Because of the importance of plasmids in many bacterial associations with plants and animals, we determined the occurrence and distribution of plasmid DNA in symbiotic Vibrio fischeri from the light organ of the sepiolid squid Euprymna scolopes. Analyses of 225 isolates of symbiotic V. fischeri from 25 individual squids revealed an overall plasmid-carriage rate of 56%. A large plasmid (greater than or equal to 39 kb) was detected in 96% of those isolates carrying plasmids, and multiple small plasmids were found to co-occur with one of the large plasmids in 81% of plasmid-carrying strains. In addition, these plasmids appear to be restricted to V. fischeri strains isolated from E. scolopes and from seawater at sites of squid populations. We were unable to assign a role or function to these plasmids, but they do not carry genes required for the establishment of the light organ symbiosis. We conclude that the essential bacterial symbiotic determinants must be encoded on the chromosome and that the plasmids may carry genes that are important for the survival of these V. fischeri strains outside of the symbiotic association.