Half of all men experience symptoms of prostatitis at some time in their lives, but the etiology is unknown for more than 90% of patients. Optimal clinical and culture methods were used to select 135 men with chronic prostatitis refractory to multiple previous courses of antimicrobial therapy. The subjects had no evidence of structural or functional lower genitourinary tract abnormalities of bacteriuria or bacterial prostatitis by traditional clinical tests, or of urethritis or urethral pathogens by culture. Specific PCR assays detected Mycoplasma genitalium, Chlamydia trachomatis, or Trichomonas vaginalis in 10 patients (8%). Broad-spectrum PCR tests detected tetracycline resistance-encoding genes, tetM-tetO-tetS, in 25% of patients and 16S rRNA in 77% of subjects. The tetM-tetO-tetS-positive cases constituted a subset of the 16S rRNA-positive cases. Patients with 16S rRNA were more likely to have >1,000 leukocytes per mm 3 in their expressed prostatic secretions than men whose prostate biopsy specimens were negative for 16S rRNA (P < 0.001). Based on direct sequencing and repetitive cloning, multiple sources of 16S rRNA were observed in individual patients. Sequences of 29 cloned PCR products revealed 16S rRNAs distinct from those of common skin and gut flora. These findings suggest that the prostate can harbor microorganisms that are not detectable by traditional approaches. These organisms may be associated with inflammation in the expressed prostatic secretions. Molecular methods hold great promise for identifying culture-resistant microorganisms in patients with chronic prostatitis.