Amplified ribosomal DNA restriction analysis (rrn-ARDRA) is based on PCR amplification and restriction of a fragment of rRNA genes including 16S and 23S genes and the intergenic spacer. rrn-ARDRA was evaluated for the identification of species within the genus Streptococcus. A total of 148 type and reference strains of pyogenic, oral, and group D streptococci were examined in order to construct a database for identification of streptococci. The amplified product was a single band approximately 4,500 bp long. This amplicon was digested separately with three (HhaI, MboII, and Sau3A) restriction endonucleases. Respectively, 27, 26, and 28 major patterns were observed after HhaI, MboII, and Sau3A restrictions. Streptococcal strains belonging to different species had different patterns or different combination of patterns. An identification system based upon a combination of the three restriction patterns in a single database was then proposed. rrn-ARDRA was successfully applied to 11 clinical isolates whose identification to the species level was difficult to obtain by phenotypic analysis. Using a database of well-characterized strains, rrn-ARDRA is a powerful method for the identification of streptococcal isolates.Streptococci are major pathogens for animals and human beings. Phenotypic studies and DNA-DNA hybridization assays have demonstrated that the genus Streptococcus may be divided in three major groups of species: pyogenic, oral, and group D streptococci (5,6,17,20,21,27).Identification of streptococcal species is currently based on observation of the cultural and morphological characteristics, determination of the biochemical pattern (production of enzymes and production of acid from various carbohydrate sources) (4,5,6,9,11,17,20), and observation of the antigenic structure according to the classification of Rebecca Lancefield (22).Molecular tools as ribotyping (3,8,25,26), oligonucleotide probing (2), PCR-based protocols (13, 23), and DNA sequence analysis (1,19,24) are now proposed to provide an accurate identification of streptococcal isolates. Among these, restriction fragment length polymorphism analysis was proposed as a general method for bacterial identification and typing (15). Amplified ribosomal DNA (rDNA) restriction analysis (ARDRA) was recently reported to be a rapid and efficient method of identification of bacterial isolates to the species level in mollicutes (7) and in some genera such as Acinetobacter or Mycobacterium (30, 31). The initial protocol was substantially improved by amplification of a larger fragment of the rrn operon (16S rRNA gene, 16S-23S intergenic spacer, and 23S rRNA gene) followed by the digestion with one to three endonucleases selected according to the bacterial genus studied (12, 18). The latest modification of this method uses a unique combination of three restriction endonucleases (HhaI, MboII, and Sau3A) irrespective of the bacterial species studied. This modified protocol was successfully used for identification of the genus and the species of strains belonging t...