Although bacterial prostatitis is a common diagnosis, well documented infections of the prostate are uncommon. Culture studies of prostate tissue led our group to hypothesize that bacterial colonization/invasion of the prostate gland might occur more commonly than is appreciated by standard microbiological techniques. Specific polymerase chain reaction (PCR) assays were used for each of the pathogens previously implicated in chronic prostatitis as well as broad-spectrum PCR assays to identify tetracycline resistance genes and bacterial ribosomal-encoding genes (16S rDNAs), followed by cloning and sequencing of the PCR products. Only ten (8%) of the 135 patients with chronic prostatitis had positive specific PCR assays including: Mycoplasma genitalium in four men, Chlamydia trachomatis in three and Trichomonas vaginalis in two, as well as one man positive for both M. genitalium and C. trachomatis. In contrast to the specific probes, the broad-spectrum PCR assays had a substantial proportion of positives. We found evidence of tetracycline resistance in 25% of patients. 16S rDNA-encoding sequences in 77% of the subjects. The tetracycline resistance positives were a subset of the 16S rDNA positive patients. Patients with 16S rDNA-encoding sequences were significantly more likely to have expressed prostatic secretion leukocytes. Many patients with chronic prostatitis/chronic pelvic pain syndrome have a wide variety of bacterial DNA-encoding sequences despite extensive negative microbiological investigations. Understanding the precise role of infection in this syndrome may well lead to better methods to elucidate the microbiology of the prostate in health and disease.