The aim of this study was to examine tetracycline-resistant gonococci and to set up a real-time PCR method to identify, in the same assay, both the chromosomally and the plasmid-mediated tetracycline-resistant genotypes. A retrospective analysis for tetracycline susceptibility was performed by the E-test and agar dilution methods on 289 gonococci isolated in Italy from 2003 to 2005. Molecular mechanisms of resistance were investigated by both sequence analyses of the three main genes associated with chromosomally mediated resistance (mtrR, penB and rpsJ genes) and by the identification of plasmids carrying the tetM determinant associated with plasmid-mediated resistance, by PCR (American- or Dutch-type plasmids). The genetic relatedness of nonsusceptible strains was evaluated by pulsed field gel electrophoresis (PFGE). The results showed the presence of 22.5% tetracycline-resistant and 49.5% tetracycline-intermediate gonococci. Coexistence of chromosomally and plasmid-mediated resistance to tetracycline was observed in the majority of resistant isolates. No clonal structure was highlighted by analysis of PFGE pattern profiles. Real-time PCR assay was able to identify all the tetracycline nonsusceptible gonococci correctly for the presence of both chromosomally and/or plasmid-mediated genotypes.