To examine a possible relationship between Chlamydia pneumoniae infection and multiple sclerosis (MS), we undertook an immunohistochemical (IHC), molecular, and ultrastructural comparison of central nervous system (CNS) tissue and cerebrospinal fluid (CSF) sediment from patients with MS and control individuals with other neurological diseases (ONDs). In 7 of 20 MS cases, IHC staining was seen in association with ependymal surfaces and periventricular regions of formalin-fixed brain tissue, by use of 3 different antichlamydial antibodies. There was no staining with any of the 3 antichlamydial antibodies in formalin-fixed brain tissue from OND controls (n=17). With available frozen CNS tissue, polymerase chain reaction (PCR) studies for the presence of C. pneumoniae genes were performed. The presence of a PCR signal was confirmed in 5 of 8 MS cases and in 3 of 18 OND controls. In an examination of CSF sediment by electron microscopy, we observed electron-dense structures resembling chlamydial organisms in CSF sediments from 11 of 20 MS cases and 2 of 12 OND controls. The presence of immunogold-labeled electron-dense bodies was correlated with the presence of a PCR signal in 10 of 11 MS cases. Results of studies using these different approaches support our suspicion of the presence of chlamydial organisms in the CNS, in a subset of patients with MS.