The replication region of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (strain P25X) was localized within a 5.9-kbp DNA fragment and its sequence was determined. An interesting feature of the sequence is the presence of a 1.3-kbp region containing seven, highly conserved, direct repeats of 72 bp in length. The pRA2 replication region has two open reading frames (ORFs). ORF1 appeared to be essential for replication and had the potential to encode a novel 30-kDa protein with a predicted helix-turn-helix motif located at the C-terminal end. ORF2 was not essential for replication and may encode for a 37-kDa protein which shares 41% and 27% amino acid sequence identity to the KfrA proteins from plasmids RK2 and R751, respectively. The essential region of replication was narrowed down to 2819 nucleotides and included four of the seven 72-bp direct repeats, a potential DnaA-binding site and ORF1.