Streptococcus sobrinus and Streptococcus downei are closely related cariogenic species. A pair of polymerase chain reaction (PCR) primers for dextranase gene of S. sobrinus NIDR6715 was designed for differentiation of S. sobrinus and S. downei. The primers amplified a single PCR product from S. sobrinus and S. downei but none of the other mutans streptococci nor other gram-positive cocci. To differentiate S. sobrinus and S. downei, a restriction analysis of the amplified products was carried out. These two species were effectively differentiated by digestion with either BamHI, BstPI, EcoRI, HaeIII, PvuII, or SalI. These results suggest that PCR amplification followed by restriction digestion is a useful method for simple and rapid differentiation of S. sobrinus and S. downei.