Mitis group streptococci are pioneer colonizers of tooth surfaces and are implicated in various pathologies. Thus, accurate identification of oral mitis group strains would be valuable for studies of plaque ecology and dental caries and for diagnostic use in endocarditis or sepsis patients. The aim of this study was to evaluate the usefulness of PCR-RFLP analysis of the 16S-23S intergenic spacer for differentiating and identifying streptococcus mitis group species. The 16S-23S rDNA spacer regions of 27 type and reference Streptococcus strains, representing 8 species, were studied by PCR-mediated amplification by using oligonucleotide primers FGPS 1490-72 and FGPL 132'-38. PCR products were digested, independently, with 14 restriction enzymes. Only AluI, MboI, CfoI, HinfI and MaeII distinguished some species, particularly AluI and CfoI, but not all the species. Eight clusters were clearly generated, corresponding to currently recognized species, but only with the addition of five ITS restriction patterns, generated by AluI + MboI + CfoI + HinfI + MaeII, then clustered by UPGMA, on a distance consensus matrix. The combination of these five ITS RFLP tests allowed a relatively conclusive genomic group differentiation of mitis group species. Despite this observation, more strains of each species will need to be analyzed, particularly clinical isolates, before arriving at general conclusions about the utility of ITS restrictions for identification of strains at the species level. An ITS PCR-RFLP-based identifying method for streptococcus mitis group species would provide significant advantages over other molecular taxonomic methods which require DNA extraction and DNA-DNA hybridization.