Lactobacillus plantarum NC13, a strain derived from the L. plantarum strain L137 isolated from a traditional fermented food produced in the Philippines, contains 9 of the 15 plasmids in the parental strain. To construct a shuttle vector between L. plantarum and Escherichia coli for genetic manipulation of L137 and its derivatives, recombinant plasmids were prepared by using the 9-plasmid DNA mixture and an E. coil vector, pBluescript LI SK+. The resultant recombinant plasmids were retransferred to L. plantarum NCL21, an NC13-derived strain cured of 3 of the 9 plasmids, and 3 recombinant plasmids were obtained. The smallest plasmid, pRN14, contained a small cryptic plasmid, pLTK2, which is one of the plasmids in L, plantarum L137. Thus, the complete nucleotide sequence of pLTK2 was determined. The pLTK2 is 2295 bp in length, and has a major open reading frame of 951bp. An encoded sequence of 317-amino acids showed extensive similarity with genes encoding replication protein (repA). A putative replication origin in pLTK2 also showed high homology to those of other gram-positive bacterial plasmids that replicate by the rolling circle mechanism. The shuttle vector pRN14 contained the erythromycin resistance gene and the ColE1 and pLTK2 replication origins. Transformation of L. plantarum strains with pRN14 by electroporation was optimized to give a transformation efficiency of 2 x 10(4) transformants/mu g plasmid. Plasmid pRN14 was stably maintained in strain NCL21, as well as in L. casei K95-5.