The gram-negative anaerobic bacterium, Fusobacterium nucleatum, is a predominant member of the human oral flora. As a major component of subgingival plaque, this bacterium has a significant impact on the ecology of the oral cavity due to its ability to adhere to many different microbial species. The objective of this study was to identify and characterize plasmids and transposons that may have the potential to be developed into tools for cloning, genetic transformation, and mutagenesis of oral isolates of F. nucleatum. Analysis of a collection of laboratory strains resulted in the identification of a homologous family of small cryptic plasmids. Plasmids within this family ranged in size from 6.0 to 6.6 kb. Eighteen percent of all strains examined (n = 74) contained DNA sequences related to the plasmids. Homologous plasmid sequences were found in strains belonging to 2 of the 3 subspecies of the bacterium. The 2 smallest plasmid species were cloned in Escherichia coli to facilitate endonuclease restriction mapping. Among the strains examined for plasmids, 5 exhibited resistance to at least 10 micrograms/ml of tetracycline. These strains, all members of the subsp. polymorphum, contained a tetracycline resistance determinant (TetM) as part of a Tn916-like integrated transposon sequence. The Tn916-like element and 1 of the plasmid species co-resided in a single strain of the bacterium. Hybridization patterns of the Tn916-like sequences were identical in all 5 tetracycline-resistant strains. However, these strains appeared to be clonally distinct based on genomic fingerprinting.