OBJECTIVE Some patients with symptomatic non-gonococcal urethritis (NGU) are negative for Chlamydia trachomatis, mycoplasmas and ureaplasmas. The optimal antimicrobial chemotherapy for such NGU has not fully been elucidated, though many studies of antimicrobial chemotherapies for C. trachomatis-positive NGU have been performed. We assessed the efficacy of antimicrobial agents that are active against C. trachomatis on non-mycoplasmal, non-ureaplasmal and non-chlamydial NGU (NMNUNCNGU). METHODS One hundred men whose first-pass urine samples were negative for C. trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum were treated with levofloxacin, gatifloxacin, minocycline, or clarithromycin for 7 days. Urethritis symptoms and the presence of polymorphonuclear leukocytes (PMNL) in urethral smears were assessed before and after treatment. RESULTS Eighty-eight (88.0%) of 100 men with NMNUNCNGU showed no signs of urethral inflammation after treatment, but two men complained of some symptoms of urethritis. Twelve (12.0%) of 100 men had significant numbers of PMNL in urethral smears, but five of these 12 men had no symptoms of urethritis. The efficacy for normalization of urethral smears was 90.7% for clarithromycin, 89.7% for levofloxacin, 87.5% for gatifloxacin, and 75.0% for minocycline. The 12 men who showed signs of urethral inflammation were retreated with levofloxacin, gatifloxacin, minocycline or clarithromycin for an additional 7 days. The 10 men who returned after the second treatment had negative urethral smears. CONCLUSIONS Our present findings suggest that antimicrobial agents active against C. trachomatis are effective against NMNUNCNGU and that a 7-day treatment regimen with an appropriate antimicrobial agent may be sufficient to manage patients with NMNUNCNGU.