To investigate the potential association between prostate infection and chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), we used molecular approaches described in previous reports. These methods employed standard polymerase chain (PCR) reaction assays to provide a qualitative evaluation of prostatic bacterial species. Here, we report on the detection of prostatic bacteria using a real-time PCR. Template DNAs were examined from prostatic tissue samples from patients with CP/CPPS. Two PCR primer sets were used: one that amplifies a portion of all known bacterial ribosomal DNAs (16S rDNAs) and one that is specific for Escherichia coli as opposed to related, E. coli-like bacteria. The 16S rDNA real-time PCR assay detected bacterial DNAs in eight (26%) of 31 samples from patients with CP/CPPS, including three samples (10%) that were also positive by the E. coli real-time PCR assay. These E. coli positives were quantified at approximately 10(3) cfu/ml of tissue digested. Quantification, speed and specificity make real-time PCR a promising approach for the quantitative detection and identification of prostatic bacteria from CP/CPPS patients.