Different parts of the Mycobacterium fortuitum plasmid pAL5000 necessary for plasmid replication and incompatibility were defined and studied. Two ORFs, named repA and repB, were defined which are necessary for replication. A pAL5000 derivative deleted in these genes can be made to replicate by providing the gene products in trans. A 435 bp fragment was defined which was necessary in cis for replication and which had an influence on copy number. This region (inc), which contains several repeated motifs, was also able to confer a degree of incompatibility when cloned into an otherwise unrelated mycobacterial replicon. pAL5000-derived plasmids carrying two copies of the inc region had a lower copy number and were less stable than the wild-type. These effects were only observed when the two regions were in the same orientation. Plasmids carrying only the inc region and no other parts of pAL5000 could be made to replicate if repA and repB were supplied in trans from another plasmid. Based on these findings, systems for selectively curing cells of one plasmid of a pair were designed and shown to be functional in Mycobacterium smegmatis. These have potential as a simple delivery system for achieving transposon mutagenesis or gene replacement in mycobacteria.