Standard identification of Streptococcus pneumoniae by optochin and bile solubility testing can lead to ambiguous results for certain isolates. Newer bacteriologic identification techniques (e.g., DNA probes) now exist. In a prospective point prevalence study of oropharyngeal S. pneumoniae carriage rates among outpatients, we compared standard organism identification techniques to DNA probe testing. By standard identification criteria, 35 (4%) of 872 isolates were characterized as presumptive S. pneumoniae. Thirty of 35 presumptive isolates were recoverable for DNA probing; 9 (30%) presumptive isolates were confirmed using a DNA probe. The antimicrobial susceptibility pattern of these DNA probe positive isolates closely paralleled that of clinical blood isolates of S. pneumoniae obtained during the study period. The 21 (70%) DNA probe negative isolates, which may represent phylogenetically related species (such as S. mitis or S. oralis), had significantly reduced antimicrobial susceptibility patterns when compared with the DNA probe positive isolates. In colonization studies, if classic criteria (optochin disc zone and bile solubility) are the sole means of identification, S. pneumoniae penicillin resistance rates may be over-reported.