Based on the availability of samples, 13 patients with culture-positive pharyngeal, urogenital, and/or anorectal gonococcal infections were selected for study in the first quarter of 1999. Of these, 3 were women (all heterosexual) and 10 were men (6 homosexual, 1 bisexual, and 3 heterosexual). The age distribution was 20 to 59 years; nine patients reported having had previous STDs. At the STD clinic, samples for gonococcal cultures were collected, directly inoculated onto GC-Lectagar plates (Becton Dickinson Biosciences, Sparks, Md.), incubated at 37°C in candle jars with moist wads of cotton, and transported to the laboratory on the same day. After incubation at 37°C for 48 h, suspected colonies were then subjected to Gram staining and identified biochemically (4). Isolates were subcultured and stored until use at −70°C in 20% (wt/vol) glycerol in microbank vials (Nunc A/S, Roskilde, Denmark). Antibiotic sensitivity testing of penicillin, tetracycline, azithromycin, and ciprofloxacin was performed by the disk diffusion method according to the recommendations of the National Committee for Clinical Laboratory Standards. Reference strains used were Moraxella catarrhalis ATCC 25238, Neisseria lactamica ATCC 23970, and Neisseria meningitidis ATCC 13090. In addition, four strains of N. gonorrhoeae known to be temporally and geographically diverse (3 Antw., 1979-Thailand; 8 Antw., 1989-United Kingdom; 18 Antw., 1989-Gambia; and 11 Antw., 1993-United Kingdom) and characterized by other typing methods were used to evaluate the AFLP genotyping analysis for N. gonorrhoeae (9). To assess the genomic stability of N. gonorrhoeae during laboratory passage, AFLP patterns were studied both before and after laboratory passage. Five strains (2 of the 22 studied and 3 single nonrelated isolates) of N. gonorrhoeae were subcultured every other day for 15 days. On days 1, 2, 4, 6, 8, and 15, chromosomal DNA was isolated by standard methods and subjected to AFLP analysis (5). There were no striking changes in the AFLP pattern of any isolate after six passages during 15 days. Thus, the AFLP patterns were conserved following in vitro passage. The 22 isolates studied were obtained from various sites: urethra (n = 10), tonsil (n = 6), rectal swabs (n = 4), and cervix (n = 2). For nine patients, two isolates from different sites could be typed. In these cases, the paired isolates were identical with regard to sensitivity to penicillin, tetracycline, azithromycin, and ciprofloxacin. This was also the case for the three pairs representing known sexual contacts (E-F, G-H, and J-L). Figure Figure11 shows the AFLP patterns of N. gonorrhoeae isolates obtained from 13 different patients. Comparison of the strains obtained from two different sites of individual patients revealed that these pairs had indistinguishable AFLP patterns. Figure Figure11 also shows that AFLP patterns of N. gonorrhoeae isolates from persons known to be sexual partners were the same within each pair. Cluster analysis showed correlation levels of >90% between the partners, irrespective of the anatomical source of the isolates. Unrelated strains from different geographic origins and different years of collection did not cluster with the presented N. gonorrhoeae isolates, nor did the ATCC reference strains. The AFLP technique appears to be reliable and reproducible, as indicated by the identical AFLP patterns of N. gonorrhoeae isolates from different sites in index patients and those of index patients and their sexual partners. Cluster analysis of isolates from the same patient but from different locations revealed AFLP patterns with 90 to 95% homology. Using standardization by defining windows of discrimination as described earlier (7), these percentages are considered to represent identical strains. The same is true for isolates from known sexual contacts. Temporally as well as geographically diverse strains showed 80 to 90% homology, indicating different strains from the same species. AFLP patterns of other members of the family Neisseriaceae showed far less homology, ranging from 40 to 50% and indicating isolates of the same genus but of different species.