Some derivatives of pIJ101, a 8.9 kb Streptomyces multi-copy plasmid, can co-exist with each other at similar copy numbers but others are strongly incompatible. The DNA sequence, sti, which causes this "strong incompatibility" was localised on a DNA segment of about 200 bp which is not part of the essential replication region of pIJ101. The sti function is active only when the DNA fragment carrying it is present in the natural orientation with respect to the basic replicon region of pIJ101. Pairs of plasmids which either both possess sti in the correct orientation (Sti+) or both lack sti or carry it in reverse orientation (Sti-) can co-exist, but Sti+ and Sti- plasmids cannot; in this case the Sti+ plasmid is retained and the Sti- plasmid is lost. This phenomenon is called strong incompatibility to distinguish it from classical incompatibility where identical or related plasmids are incompatible and dissimilar plasmids are compatible. pIJ101 probably replicates via a single-stranded intermediate; sti would be a site where the synthesis of the second (lagging) DNA strand is initiated because Sti- plasmids accumulate more single-stranded plasmid DNA than Sti+ plasmids. The copy number of pIJ101 and its derivatives is influenced by sti and by an additional trans-acting function (cop).