Only two plasmids have been isolated and characterized from the entire genus Mycoplasma, which includes over 90 recognized species. Both of these plasmids were obtained from the same species. Mycoplasma mycoides subsp. mycoides. We have previously characterized one of these plasmids, pKMK1, as a preliminary step in developing mycoplasmal cloning vectors. In the present study, we have separately combined pKMK1 with two different Escherichia coli replicons and a tetracycline resistance (tetM) gene. One of the constructs, plasmid p2D4, was shuttled from E. coli to M. mycoides subsp. mycoides and back to E. coli with no deletions or rearrangements occurring in the plasmid. In the second construct, the E. coli replicon was deleted when the plasmid was transformed into M. mycoides subsp. mycoides. This derivative, designated plasmid pIKΔ, is noteworthy in that it could be transformed into M. mycoides subsp. mycoides at a much higher frequency than the parental plasmid. A gram- positive bacterial erythromycin resistance determinant (erm) was cloned into both p2D4 and pIKΔ. Resistance to erythromycin was stably maintained using both constructs, even in the absence of erythromycin selection, indicating that these plasmids will be useful mycoplasmal cloning vectors. © 1994 Academic Press, Inc.