The SaPIs are 14- to 17-kb mobile pathogenicity islands in staphylococci that carry genes for superantigen toxins and other virulence factors and are responsible for the toxic shock syndrome and other superantigen-related diseases. They reside at specific chromosomal sites and are induced by certain bacteriophages to initiate an excision-replication-packaging program, resulting in their incorporation into small infective phage-like particles. These are responsible for very high transfer frequencies that often equal and sometimes exceed the plaque-forming titer of the inducing phage. The ability of the SaPIs to replicate autonomously defines them as individual replicons and, like other prokaryotic replicons, they possess replicon-specific initiation functions. In this paper, we report identification of the SaPI replication origin (ori) and replication initiation protein (Rep), which has helicase as well as initiation activity. The SaPI oris are binding sites for the respective Rep proteins and consist of multiple oligonucleotide repeats in two sets, flanking an AT-rich region that may be the site of initial melting. Plasmids containing the rep-ori complex plus an additional gene, pri, can replicate autonomously in Staphylococcus aureus but are very unstable, probably because of defective segregation.