The region of the 29.5-kb plasmid pSX267 from Staphylococcus xylosus DSM 20267 that is required for autonomous replication in staphylococci was isolated on a 1.8-kb DNA fragment. The sequence analysis of the fragment yielded two open reading frames, repA and orf2, encoding proteins of 37.2 and 13.2 kDa, respectively. The deduced amino acid sequence of repA showed similarity to the replication initiator protein of plasmid pAD1 from Enterococcus faecalis, to two proteins of unknown function encoded by the E. faecalis plasmid pCF10 and the lactobacillus helveticus plasmid pLJ1, and surprisingly to the mouse interferon-response element binding factor 1. The repA gene of pSX267 is indispensable for replication suggesting that it encodes the replication initiator protein of pSX267. Introduction of a frameshift mutation into repA or deletion of 26 codons at its 3'-end resulted in nonreplicative plasmids. Removal of sequences required for repA expression also abolished replication. Complementation experiments with repA in trans identified the ori of pSX267 within the repA coding region. The second orf, which is located downstream of repA, could be deleted without affecting plasmid replication. It seems to be a nonfunctional remainder of a rolling circle plasmid of the pC194/pUB110 family, since its deduced amino acid sequence resembles the pC194/pUB110-type replication proteins. The minimal replicon of pSX267 defined so far comprises 1255 bp. Besides repA, no other plasmid-encoded gene is required to mediate replication in staphylococci.