We have developed a duplex real-time PCR for the rapid diagnosis of Streptococcus pneumoniae infection from culture-negative clinical samples with the simultaneous determination of penicillin susceptibility. The assay amplifies a lytA gene target and a penicillin binding protein 2b (pbp2b) gene target in penicillin-susceptible organisms. The assay was shown to be sensitive (detects 0.5 CFU per PCR) and specific for the detection of S. pneumoniae DNA. The assay was validated by comparing pbp2b PCR results with MIC data for 27 S. pneumoniae isolates. All 5 isolates with penicillin MICs of >1.0 mg/liter were pbp2b real-time PCR negative, as were 9 of the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter. One isolate with a penicillin MIC of 0.12 to 1.0 mg/liter gave an equivocal pbp2b real-time PCR result. Twelve isolates were penicillin susceptible (MICs of <0.06 mg/liter) and pbp2b real-time PCR positive. These data were used to establish an algorithm for the interpretation of penicillin susceptibility from the duplex PCR result. pbp2b real-time PCR results were also compared to an established PCR-restriction fragment length polymorphism (RFLP) method previously applied to these 27 isolates and 46 culture-negative clinical samples (containing S. pneumoniae DNA by broad-range 16S rRNA gene PCR). Discordant results were seen for four isolates and six culture-negative clinical samples, as PCR-RFLP could not reliably detect penicillin MICs of 0.12 to 1.0 mg/liter. We report prospective application of the duplex PCR assay to the diagnosis of S. pneumoniae infection from 200 culture-negative clinical specimens sent to the laboratory for diagnostic broad-range 16S rRNA gene PCR. One hundred six were negative in the duplex PCR. Ninety-four were lytA PCR positive, and 70 of these were also pbp2b PCR positive and interpreted as penicillin susceptible. Fourteen were pbp2b PCR negative and interpreted as having reduced susceptibility to penicillin. For the remaining 10 samples, susceptibility to penicillin was not determined.