Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.