The cryptic plasmid pSBO1 (3904 bp) was isolated from Streptococcus bovis JB1. pSBO1 contained an open reading frame (ORF) that is homologous to sequences encoding the replication protein (Rep) in pEFC1 (isolated from Enterococcus faecalis), pSK639 (Staphylococcus epidermidis), pLA103 (Lactobacillus acidophilus), and pUCL287 (Tetragenococcus halophila). In addition, four 22-bp direct repeats (DRs) were located upstream of the putative replication gene (rep) of pSBO1. Recombinant plasmids (pSBE10 and pSBE11) containing the DRs and putative rep of pSBO1 replicated in S. bovis 12-U-1 and no8 strains. This result indicates that the putative rep encoded Rep and that the replicon of pSBO1 contained the DRs and the rep. Gel shift assays showed that the Rep of pSBO1 bound the 22-bp DRs.