The Public Health Laboratory Network (PHLN) convened a workshop of Australian experts in Melbourne on 23 March 2005 to identify laboratory issues of relevance and suggest guidelines for use of nucleic acid detection tests (NADT) for diagnosis of gonorrhoea in Australia. The proceedings of that meeting were endorsed by the members of the PHLN and the Communicable Diseases Network of Australia. Given the present state of knowledge and experience of conditions currently existing in Australia, the following recommendations were made: Recommendation 1: Assays using detection of the cppB gene should not be used for either screening or supplemental assays. Recommendation 2: All in-house screening assays that are positive should also be positive on a reliable supplemental assay before a positive result is reported. Recommendation 3: All commercial screening assays that are positive should also be positive on a reliable supplemental assay before a positive result is reported. Recommendation 4: If a sample is positive in a screening assay but a suitable supplemental assay is negative, then the result should be reported as negative. Recommendation 5: Laboratories should ensure that the test combination they use would yield a positive predictive value of at least 90 per cent in a population with a prevalence of 1 per cent. Recommendation 6: For the purposes of test evaluation, as distinct from diagnostic testing, true positives be defined by meeting one or more of the following criteria: 1) culture positive using contemporary isolation and identification techniques; 2) positive result on NADTs directed to targets on three separate genes that are known to have discriminatory capacity; 3) sequencing of a gene known to separate gonococcal from non-gonococcal species. Recommendation 7: Inhibitor controls should be routinely included in all NADT. Recommendation 8: Cultures are the preferred test for samples from non-genital sites. If however it is necessary to perform a NADT, then more stringent criteria should be applied, and positive samples should meet the 'test evaluation' criteria for a 'true positive'. Recommendation 9: In order to properly assess the routine diagnostic system in Australia, the following quality assurance samples should be distributed in addition to the routine samples currently used: 1. cppB negative N. gonorrhoeae; 2. Non-gonococcal species known to cause false positive reactions: these should be dispatched both as a single species, as well as mixture with N. gonorrhoeae. In the latter circumstance, the non-gonococcal species should be present in 10-fold excess; 3. Urine samples: preferably a single patient sample, otherwise a spiked sample. 4. Validation panels should be made that include samples that are culture positive but PCR negative. True positive samples should also be made available. In addition, a process should be established for full phenotypic and genotypic characterisation of unidentified species that yield false positive results in NADT for gonococci. Recommendation 10: Strategies should be put in place to ensure that sufficient numbers of gonococcal isolates are obtained to allow reliable monitoring of antimicrobial resistance. Recommendation 11: Public health practitioners need to define the relevant populations that need to be targeted and identify any that require enhanced surveillance.