Eight identification methods were evaluated against 100 isolates of Neisseria gonorrhoeae and 21 non-gonococcal Neisseria strains. The methods examined included four commercial biochemical kits, API NH, RapID NH, Gonochek II and Neisseria Preformed Enzyme Test (PET), three immunological kits, Phadebact Monoclonal GC test, GonoGen II and MicroTrak, and one in-house carbohydrate-utilization method, cystine trypticase agar (CTA) sugars. The percentage of isolates unambiguously identified as N. gonorrhoeae by each of the methods was as follows: API NH, 66%; RapID NH, 64%; GonoChek II, 66%; Neisseria PET, 66%; Phadebact Monoclonal GC OMNI test, 99%; GonoGen II, 100%; MicroTrak, 100%; and CTA sugars, 96%. The low sensitivity of the biochemical kits for the identification of N. gonorrhoeae was due to a lack of the enzyme proline iminopeptidase (Pip) in 34% of the isolates examined. All the biochemical kits utilized the presence of this enzyme as a marker for N. gonorrhoeae. The Phadebact Monoclonal GC kit, GonoGen II, MicroTrak, CTA sugars and the API NH kit all exhibited high specificity, but non-gonococcal Neisseria were misidentified as N. gonorrhoeae using RapID NH (two strains), Gonochek II (11 strains) and Neisseria PET (11 strains). Whilst the isolates examined in this study may not be truly representative, they do indicate that N. gonorrhoeae isolates lacking the enzyme Pip can give anomalous results when using commercially available biochemical tests and that some non-pathogenic Neisseria species are still being misidentified using some biochemical kits. This further reinforces the recommendation that any dubious biochemical result should be confirmed with an immunological test.