The activity of temafloxacin hydrochloride was evaluated by agar dilution against 100 clinical isolates of Neisseria gonorrhoeae and compared with the activities of penicillin, tetracycline, ceftriaxone, ciprofloxacin, and ofloxacin. Temafloxacin inhibited 100% of study isolates at a concentration of 0.015 microgram/ml or less and was highly active against penicillin- and tetracycline-resistant strains. The in vitro activity of temafloxacin was nearly identical to that of ceftriaxone and was slightly less than that observed with ciprofloxacin and ofloxacin. Temafloxacin represents a promising alternative agent for investigation in the treatment of infection due to N. gonorrhoeae.