The nucleotide sequence of a region of plasmid RSF 1030 that includes the origin of DNA replication was determined using the DNA of a small derivative, pST19. The nucleotide sequence of the pST 19 origin region is very similar to that of the ColE1 origin except for a 25 base pair (bp) deletion about 350 bp upstream of the origin and a considerable difference in the region between 400 and 600 bp upstream of the origin. Replication of pST19 starts at one of three consecutive nucleotides (dA, dA or dC) located at a unique position in the region where the nucleotide sequence is identical to that of the ColE1 origin. There are two major sites of initiation of transcription in the region. Transcription from one of the sites yields the primer precursor that can be cleaved by RNase H to form the primer of about 530 nucleotides long. Transcription from the other site proceeds on the opposite strand and terminates close to the primer initiation site to yield species I RNA (or RNA I) about 105 nucleotides long. The presumed RNA polymerase binding sites in the promoters of these transcripts differ from those of the corresponding ColE1 transcripts. Incompatibility specified by pST19 is different from that specified by ColE1. Hypothetical peptides encoded by the origin region of these plasmids are unlikely to be involved in the determination of incompatibility. It has been shown that RNA I is an incompatibility-group specific inhibitor of primer formation. Despite a significant difference in nucleotide sequence, the primer RNA and RNA I of pST19 can be folded into structures analogous to those of the ColE1 transcripts.