BACKGROUND Strains of Neisseria gonorrhoeae resistant to penicillin and ciprofloxacin have been isolated worldwide. Increasing number of N. gonorrhoeae that lack the enzyme proline aminopeptidase (proA-negative N. gonorrhoeae) have been detected in many countries all over the world. OBJECTIVE This study aims to assess the resistance profiles of N. gonorrhoeae isolates sent to the Department of Clinical Microbiology in Vejle, Denmark, between 2003 and 2007, and to analyse their biochemical patterns. METHODS Sixty-two strains of N. gonorrhoeae were retrospectively analysed for their susceptibility to penicillin, ciprofloxacin and ceftriaxone. The identification of isolated strains was confirmed using both biochemical and immunological tests. RESULTS Twenty-one (33.9%) N. gonorrhoeae isolates were resistant to penicillin and 30 (48.4%) were resistant to ciprofloxacin. All strains were susceptible to ceftriaxone. Fifty-six (90.3%) N. gonorrhoeae strains showed API NH biochemical code 10,010 (produced acid from glucose and proline aminopeptidase). Six strains showed code 10,000 that lack the enzyme proline aminopeptidase (proA-negative N. gonorrhoeae). CONCLUSIONS Ceftriaxone should be used as the first-line treatment of gonorrhoea in Vejle community area, Denmark, both for infections with proA-producing and proA-negative N. gonorrhoeae isolates, which circulate in the region.