Characterization of an extrachromosomal element from an organism in the genus Spiroplasma is likely to be an essential step in the development of cloning vectors which replicate in these organisms. A restriction map for an II-kb element, designated pCT-I, isolated from Spiroplasma taiwanense train CT-I (ATCC 43302) has been constructed using the restriction enzymes Bg /II, EcoRI, HincII, HindIII, HpaI, Pst I, and XhaI. This element is distinct from any previously characterized spiroplasma virus or plasmid. pCT-1 has been cloned into the Escherichia coli vector pBR322 as a step in the development of a biphasic shuttle vector system.