Chlamydophila pneumoniaeis an intracellular pathogen responsible for respiratory tract infections. The isolation of the microorganism from clinical specimens is essential for a diagnosis. However, the identification ofC. pneumoniaeby cell cultures is very difficult besides strongly depending on the sample conditions. The study aimed to investigate, in adult patients with pharyngotonsillitis, the frequency ofChlamydophila pneumoniaedetection by cell cultures and three conventional PCRs (a conventional PCR targeting the 16S rRNA gene and two nested PCRs, targeting the 16S rRNA gene and the ompA gene, respectively). The presence of chlamydial inclusion in cell cultures was observed in 11/94 samples (11.70%) by IFA.C. pneumoniaeDNA was detected in 12/94 (12.76%) specimens by the 16S rRNA gene nested PCR, 4/94 (4.26%) by ompA gene nested PCR, and in 2/94 (2.13%) by 16S rRNA single-step PCR. Our data show poor agreement between the three applied DNA-amplification methods; in fact, only 16S rRNA gene nested PCR showed a statistically significant difference. Moreover, this result allowed us to achieve a definitive confirmation of the previous finding and to avoid the risk of an overestimation of theC. pneumoniaeas a pathogen in pharyngotonsillitis.