We report on the case of a 54-year-old woman diagnosed as having culture-negative endocarditis (clinical and histopathologic evidence compatible with a recent episode of endocarditis). The responsibility of Chlamydia pneumoniae in this episode of endocarditis was suggested by a serological study and was then confirmed by the positive results of PCR and in situ hybridization tests with aortic and mitral valves tissues. To our knowledge, this is the first case of endocarditis due to C. pneumoniae confirmed by molecular biology-based techniques.