When testing 248 clinical isolates of Neisseria gonorrhoeae, the sensitivity was 100% with GonoGen (Becton Dickinson Microbiology Systems), 99.6% (247 of 248) with GonoGen II (Becton Dickinson), 97.2% (241 of 248) with the MicroTrak direct fluorescent-antibody test (Syva), and 97.6% (242 of 248) with Rapid Fermentation Agar carbohydrates (Remel). Of 62 isolates of other Neisseria species, none was misidentified as N. gonorrhoeae by GonoGen, MicroTrak, or Rapid Fermentation Agar carbohydrates but 7 (31.8%) of 22 isolates of N. meningitidis gave strong, repeatedly false-positive results with GonoGen II. The sensitivity of all four assays was good to excellent, but all positive GonoGen II results should be confirmed with an independent assay, especially when isolates are recovered from sites where N. meningitidis is likely. Positive results from any of the assays should be routinely confirmed when dictated by specific clinical, legal, or microbiological circumstances.