A 1.6-kb DNA region required for the replication of pSW500 from Erwinia stewartii SW2 has been identified. DNA sequencing analysis revealed that this DNA fragment consists of a DnaA box, seven 16-bp direct repeats, and a 1005-bp open reading frame. The seven direct repeats have been demonstrated to mediate the incompatibility function of the plasmid. Primer extension analysis showed that the 1005-bp ORF is transcribed in vivo and the +1 site of the transcript is located 113 bp upstream from the translation initiation codon of the ORF. Complementation studies showed that this ORF is required for the replication of the plasmid and may encode a replication protein, RepA. Gene fusion studies revealed that the expression of repA is autoregulated by RepA. We also found that the pSW500 replicon has a copy number of approximately two and that the plasmid is stably maintained in Escherichia coli, thus demonstrating that the replicon contains all the elements required for copy number control and plasmid stability in E. coli. Curing of pSW500 from E. stewartii SW2 revealed that loss of pSW500 did not have any obvious effect on morphology or physiology of the cells, suggesting that pSW500 does not encode a function that is indispensable for the survival of the organism.