The genetic and molecular nature of the Ent plasmids that determine enterotoxin biosynthesis in strains of Escherichia coli isolated from man and domestic animals has been investigated. At least one transmissible plasmid was present in 90% of 96 toxigenic strains compared with 36% of 204 nontoxigenic strains. Ent plasmids were demonstrated in 17% of the toxigenic strains and often appeared to be self-transmissible. A single Ent plasmid was isolated from 15 enterotoxigenic strains by conjugation experiments. The class of Ent plasmids that determines the production of both heat-stable and heat-labile enterotoxins was homogeneous and consisted of a single DNA species with an approximate molecular weight of 6.0 × 107 daltons. The class of Ent plasmids that codes for heat-stable enterotoxins only was heterogeneous and consisted of a single species of DNA that ranged in size from 2.1 to 8.0 × 107 daltons.