A 3.6-kb plasmid, designated pND324, was isolated from Lactococcus lactis subsp. lactis LL57-1. Sequence analysis revealed the presence of three open reading frames, rep324, orfX1 and orfX2, which are flanked by two non-coding regions, ori324 and cisE. The minimal replication region of pND324 consists of ori324 and rep324, which is closely related to the lactococcal theta-type replicons of the pWV02/pCI305 family. pND324 was stable at both 30 degrees C and 37 degrees C, whereas derivatives that lack cisE were highly unstable at 37 degrees C, indicating that cisE is essential for thermostability. Sequences that are similar to orfX1 are commonly present in the lactococcal theta-type plasmids. The orfX2 product is homologous to TrfA, a 43-kDa protein of the E. coli theta-type plasmid RK2 required for replication and maintenance. Plasmid deletion and stability analyses showed that orfX2 is involved in the thermostability of pND324. Based on the minimal replication region of pND324? an integrative cloning vector, designated pND421, was constructed. In L. lactis LM0230, cells that carried pND421 integrated into its host chromosomal DNA could be recovered readily following incubation at 37 degrees C for 40 generations. The integrated plasmid was totally stable for at least 100 generations without selection at 30 degrees C.