I dentification of ␣-hemolytic streptococci is clinically important (3,15,26,27,33,34). The aim of this study was to evaluate and compare the reliability of API 20 Strep, the automated BD Phoenix system, and MALDI Biotyper MS for clinically identifying significant isolates of ␣-hemolytic streptococci to the species and group levels. MALDI Biotyper has been found to give high-confidence identifications for -hemolytic streptococci (8). As a gold standard, 16S rRNA gene sequencing was used (4,5,7,15,22,31), with a sequence homology of 99% or greater with published species sequences considered to denote the same species, in accordance with CLSI guidelines (10). For Streptococcus mitis group species, better discrimination was obtained by sequence analysis of the 16S-23S rRNA intergenic spacer (ITS) region (7). Taxonomy and nomenclature followed that outlined by Facklam at the CDC's Streptococcal Laboratory, Atlanta, GA (15), and subsequently cited and expanded upon by others (11, 24). Unlike previous studies of phenotypic methods (5,6,12,16,19,20), this was a direct comparison of phenotypic methods versus the gold standard.Bacterial isolates. Forty-nine clinically relevant isolates (40 blood culture, 2 cerebrospinal fluid (CSF), 3 urine, 1 each from ascitic fluid, joint fluid, and an infected wound, and 1 from an unspecified site but referred as clinically significant) of ␣-hemolytic streptococci (except Streptococcus pneumoniae) were collected prospectively from sterile sites cultured in three clinical laboratories. Additionally, a selection of fully characterized strains was used, including NCTC 11086 (Streptococcus sanguinis), NCTC 11427 (Streptococcus parasanguinis), NCTC 10904 (S. sanguinis), M99 and SK12 (both Streptococcus gordonii) (23), SK36 (S. sanguinis) (35), and UA159 (Streptococcus mutans) (1).API 20 Strep and BD Phoenix automated identification. API 20 Strep (bioMérieux) and BD Phoenix (Becton, Dickinson) tests were carried out according to the manufacturers' instructions, with Apiweb 1.2.1 software used to identify API codes. BD Phoenix SMIC/ID streptococcal identification panels were used. Following the manufacturers' recommendations, identification with a score of Ͻ80% for API 20 Strep or Ͻ90% for BD Phoenix was considered unacceptable. If a similar result was obtained on repeat testing, the strain was assigned as unidentified by that method.MALDI Biotyper identification. The MALDI Microflex (matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS]; Bruker Daltonics GmbH, Bremen, Germany) with MALDI Biotyper software 2.0 was used. Identifications were performed according to the manufacturer's instructions, using the full extraction procedure as described previously (18) with isolates from chocolate blood agar (Oxoid, Basingstoke, United Kingdom). The extraction method is superior to direct colony testing for Gram-positive cocci (2). Samples on the target plate were left to air dry before adding the matrix solution (saturated solution of alpha-4-cyano-hydrocycinnam...