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The aim of the present study was to determine prospectively the antimicrobial susceptibility of Neisseria gonorrhoeae strains collected in southern Germany (Heidelberg and Stuttgart areas). Sixty-five N. gonorrhoeae strains, isolated between July 2004 and June 2005 from patients with uncomplicated gonorrhoea, were tested. Minimum inhibitory concentrations of penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, ceftriaxone, and cefixime were determined by the E test. All isolates were fully susceptible to ceftriaxone, cefixime, and spectinomycin. However, 21.5% (14/65), 29.2% (19/65), and 47.7% (31/65) of isolates were resistant to penicillin (>2.0 mg/l), tetracycline (>2.0 mg/l), and ciprofloxacin (>1.0 mg/l), respectively. Critical MICs of azithromycin (>1.0 mg/l, as defined by the Neisseria Reference Laboratory at the Centers for Disease Control) were found for five (7.7%) N. gonorrhoeae isolates. These data indicate a high prevalence of N. gonorrhoeae strains resistant to the antimicrobial agents currently used to treat gonococcal infections in the Heidelberg and Stuttgart areas. Even though the findings may not be representative of the general population in Germany, they nevertheless illustrate the need to establish an antimicrobial resistance surveillance system in order to control gonorrhoea effectively.