The regulatory region of the Mycobacterium fortuitum plasmid pAL5000 was studied in vivo and in vitro by mutational analysis. This region comprises the origin of replication for the plasmid and the start point of transcription for the repA/B genes, which encode the two replication proteins RepA and RepB. In this region there are two binding sites for RepB: a low-affinity site which is probably the origin of replication and a high-affinity-site which overlaps the promoter and implies an autoregulated expression of RepB. The high-affinity site contains two 8 bp palindromes, as well as an inverted repeat structure. By introducing point mutations into each of these motifs and monitoring changes to RepB binding in a gel-retardation assay, it was shown that the central, GC-rich palindrome (the GC-box) is the most important motif for protein binding. Mutations in the second, AT-rich palindrome (the AT-box) had no effect on protein binding and the inverted repeat structure per se was not needed, though some single-base changes affected binding to one or other of the DNA strands. These mutations were subsequently tested in vivo for their effects on plasmid replication in Mycobacterium smegmatis. Any change to the GC-box abolished replication, but changes to the other motifs were dependent on the position of the changed base, again indicating that the inverted repeats are not essential and that the AT-box is part of the promoter and not primarily recognised by RepB. The mutated plasmids did not show any changes in copy number to that of the wild-type. The expression of RepB was boosted by introducing a stronger promoter upstream of the repA/B genes. The resulting plasmid was capable of increasing to a degree in trans the copy number of other plasmids carrying the ori region, but was unstable when present on its own in M.smegmatis.