An 18 633 bp region containing the replicon from the approximately 53 kb pBM400 plasmid of Bacillus megaterium QM B1551 has been sequenced and characterized. This region contained a complete rRNA operon plus 10 other potential open reading frames (ORFs). The replicon consisted of an upstream promoter and three contiguous genes (repM400, orfB and orfC) that could encode putative proteins of 428, 251 and 289 amino acids respectively. A 1.6 kb minimal replicon was defined and contained most of repM400. OrfB was shown to be required for stability. Three 12 bp identical tandem repeats were located within the coding region of repM400, and their presence on another plasmid caused incompatibility with their own cognate replicon. Nonsense, frameshift and deletion mutations in repM400 prevented replication, but each mutation could be complemented in trans. RepM400 had no significant similarity to sequences in the GenBank database, whereas five other ORFs had some similarity to gene products from other plasmids and the Bacillus genome. An rRNA operon was located upstream of the replication region and is the first rRNA operon to be sequenced from B. megaterium. Its unusual location on non-essential plasmid DNA has implications for systematics and evolutionary biology.