The replication region of the phage resistance plasmid pCI528 from Lactococcus lactis subsp. cremoris UC503 was localised to within a 10-kb HindIII restriction fragment. A 6.3-kb BglII-HindIII subclone of this fragment, cloned into a replication probe vector, allowed replication in Lactococcus but not in Bacillus or Lactobacillus. Sequence analysis revealed an ORF of 1152 bp preceded by a putative ori region containing a 22-bp sequence tandemly repeated three and three-quarter times, a second smaller direct repeat and two inverted repeats. Extensive homology was observed with the well characterised replication region of the small cryptic plasmid pCI305 (Hayes, F., Vos, P., Fitzgerald, G.F., deVos, W. and Daly, C. Plasmid 25, 16-26).