Native plasmid of Streptococcus thermophilus ST137, pER371 (2.7 kb) linearized at various unique restriction sites was individually subcloned into Escherichia coli plasmid pUC19 to generate the pUER-series recombinants. A selection cassette consisting of chloramphenicol- and erythromycin-resistance genes was spliced into each construct to generate the pMEU shuttle vectors. Electrotransformation of Streptococcus thermophilus with these vectors showed that a ca. 1.7 kb BstEII/BanII fragment is essential for plasmid replication. A shuttle vector, pMEU14’-1 (5.3 kb), was constructed using the minimally required fragment for replication. A chloramphenicol acetyltransferase (cat) gene was successfully expressed in the ultimate S. thermophilus host by using pMEU14’-1 Cloning vectors derived from pER371 should provide valuable alternative gene delivery vehicles for the genetic engineering of lactic acid bacteria.