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Microbial agents may play a role in the pathogenesis of multiple sclerosis (MS). C. pneumoniae has been recently associated with MS; however, study results are at variance. We tested the hypothesis that Chlamydia pneumoniae-specific DNA and RNA are more often detected in cerebrospinal fluid (CSF) of patients with multiple sclerosis than patients with other neurological diseases (OND). We investigated CSF samples from 84 patients with definite MS and 89 OND patients (n = 62 with normal CSF; n = 27 with pathological CSF) using a nested polymerase chain reaction (PCR) to detect ompA gene sequences of C. pneumoniae. In subjects with positive PCR, we probed for chlamydial heat shock protein 60-mRNA and 16S-rRNA by reverse transcriptase (rt)-PCR.C. pneumoniae-specific DNA was more often detected in MS patients (50 %) than in all OND patients combined (28.1%, p = 0.003) and in OND patients with normal CSF (24.2%, p = 0.003) but not than in OND patients with pathological CSF (37%, p = 0.24). In relapsing-remitting MS (n = 55), the prevalence of C. pneumoniae DNA was higher (66.7 %) than in both OND subgroups (p ≤ 0.05). In MS patients (n = 20), chlamydial heat shock protein 60-mRNA (75%) and 16S-rRNA (70%) were more often detected than in OND patients (n = 16; 18.8%; p < 0.005).Although more often detected in remitting-relapsing MS, C. pneumoniae DNA in CSF is not specific for MS owing to its high prevalence in OND controls. However, the higher rate of gene transcription suggests a more active metabolism of C. pneumoniae in MS patients.