Plasmids have rarely been detected in organisms constituting the genusMycoplasma. Recently, the isolation of a cryptic plasmid fromMycoplasma mycoides subsp.mycoides has been described, and we report here the isolation of a second cryptic plasmid from this species. Restriction map and Southern blot analyses show that the second plasmid is distinct from the previously described plasmid, although a limited region of homology was detected. The availability of mycoplasmal cryptic plasmids may lead to the development of cloning vectors that replicate in these organisms. © 1990 Academic Press, Inc. All rights reserved.