The recent emergence of a decreased susceptibility of Neisseria gonorrhoeae strains to penicillin in New Caledonia has lead clinicians to operate a change in the treatment strategy. In addition, this important health issue has emphasized the need for a rapid means of detecting penicillin resistance in N. gonorrhoeae in order to select an effective treatment and limit the spread of resistant strains. In recent years, the use of fluorescence resonance energy transfer on the LightCycler has proven to be a valuable tool for the screening of mutations occurring in the genome of various microorganisms. In this study, we developed a real-time PCR assay coupled with a fluorometric hybridization probes system to detect a penicillin resistance-associated mutation on the N. gonorrhoeae ponA gene. Following an extensive evaluation involving 136 isolates, melting curve analysis correctly evidenced a 5 degrees C T(m) shift in all N. gonorrhoeae strains possessing this mutation, as determined by conventional sequencing analysis. Moreover, the mutation profiles obtained with the real-time PCR showed good correlation with the pattern of penicillin susceptibility generated with classical antibiograms. Overall, our molecular assay allowed an accurate and reproducible determination of the susceptibility to penicillin corresponding to a mutation present in all chromosomally mediated resistant strains of N. gonorrhoeae.