We assessed whether the intracellular bacterium Chlamydia pneumoniae was present in post-mortem brain samples from patients with and without late-onset Alzheimer's disease (AD), since some indirect evidence seems to suggest that infection with the organism might be associated with the disease. Nucleic acids prepared from those samples were screened by polymerase chain reaction (PCR) assay for DNA sequences from the bacterium, and such analyses showed that brain areas with typical AD-related neuropathology were positive for the organism in 17/19 AD patients. Similar analyses of identical brain areas of 18/19 control patients were PCR-negative. Electron- and immunoelectron-microscopic studies of tissues from affected AD brain regions identified chlamydial elementary and reticulate bodies, but similar examinations of non-AD brains were negative for the bacterium. Culture studies of a subset of affected AD brain tissues for C. pneumoniae were strongly positive, while identically performed analyses of non-AD brain tissues were negative. Reverse transcription (RT)-PCR assays using RNA from affected areas of AD brains confirmed that transcripts from two important C. pneumoniae genes were present in those samples but not in controls. Immunohistochemical examination of AD brains, but not those of controls, identified C. pneumoniae within pericytes, microglia, and astroglia. Further immunolabelling studies confirmed the organisms' intracellular presence primarily in areas of neuropathology in the AD brain. Thus, C. pneumoniae is present, viable, and transcriptionally active in areas of neuropathology in the AD brain, possibly suggesting that infection with the organism is a risk factor for late-onset AD.