Detection and quantification of Mycoplasma genitalium were evaluated in 83 patients with urethritis (group 1), 60 patients with urethral symptoms but no urethritis (group 2), and 50 asymptomatic men (group 3). Quantification of M. genitalium was carried out using real-time polymerase chain reaction (PCR) analysis of first-pass urine samples. The rate of detection of M. genitalium was significantly higher in group 1 than in groups 2 and 3 (P<.0001). The mean observed concentration of M. genitalium was 1.2x10(4) equivalent genomes/mL of urine (range, 50 to 8x10(4) equivalent genomes/mL). Analysis of M. genitalium load in serial urine samples collected before and after the administration of antibacterial treatment showed an association between clinical and microbiological responses, with a shift to negative PCR results in symptom-free patients. Our results illustrate the usefulness of monitoring the M. genitalium load in evaluating the susceptibility of M. genitalium to antibacterial treatment.