Background: Streptococcus mutans and Streptococcus sobrinus are considered to be important bacterial species in the initiation of human dental caries. Therefore, the establishment of a reliable genotyping method to distinguish S. mutans from S. sobrinus is of central importance. Objective: We assessed the usefulness of repetitive extragenic palindromic polymerase chain reaction (rep-PCR) using ERIC primer banding patterns in differentiating S. mutans and S. sobrinus. Design: Five S. mutans and two S. sobrinus prototype strains and 50 clinical isolates (38 S. mutans serotype c, 4 S. sobrinus serotype d, and 8 S. sobrinus serotype g) were examined. The banding patterns of amplicons generated were compared among the prototype strains and clinical isolates, to find common bands that distinguish S. mutans and S. sobrinus. Results: Multiple banding patterns were seen with all strains tested. The representative strains of S. mutans tested revealed six unique, strong bands at 2,000 bp, 1,700 bp, 1,400 bp, 1,100 bp, 850 bp, and 250 bp, whereas S. sobrinus had seven strong bands at 2,000 bp, 1,800 bp, 1,100 bp, 900 bp, 800 bp, 600 bp, and 550 bp. The band at 1,100 bp was the only band that was observed in both S. mutans and S. sobrinus. Furthermore, most clinical S. mutans isolates revealed identical banding patterns. All S. mutans had amplicons at 1,700 bp, 850 bp, and 250 bp, whereas those of S. sobrinus were at 1,100 bp, 900 bp, and 800 bp. Conclusions: These results indicate that using rep-PCR with the ERIC primers can distinguish between S. mutans and S. sobrinus. © 2011 Tamami Okada et al.