A shuttle vector composed of pBL1, a 4.46-kb cryptic plasmid from coryneform bacterium Brevibacterium lactofermentum, and Escherichia coli vector pHSG298 was found to inhibit growth and cause cell filamentation in E coli. After construction of several deletions of pBL1, a 1.23-kb AccI-HindIII fragment was found responsible. DNA sequence analysis showed that this fragment contained a 726-bp-long open reading frame (ORF3), encoding a protein with 242 amino acid residues. Corresponding to ORF3, a 28-kDa protein was detected in an in vitro protein synthesis system. ORF3 was dispensable for the stable maintenance of pBL1 in coryneform bacteria.