Chlamydia pneumoniae has been associated with atherosclerotic cardiovascular disease by both seroepidemiologic studies and direct detection of the organism in atherosclerotic plaque by electron microscopy (EM), immunocytochemistry (ICC), and polymerase chain reaction (PCR). Despite the frequent detection of the organism in atheromatous cardiovascular specimens by these methods, only 1 cardiovascular isolate of C. pneumoniae, obtained from a coronary artery, has been previously reported. This study reports the isolation of C. pneumoniae from a prospectively obtained carotid endarterectomy specimen. The organism appears to be identical to other C. pneumoniae isolates by EM morphology, reactivity to species-specific monoclonal antibodies, and Southern hybridization analysis of chromosomal digests using C. pneumoniae-specific DNA probes. C. pneumoniae was detected by PCR or ICC (or both) in 11 (69%) of 16 other endarterectomy specimens tested by both of these methods. These results provide further evidence for an association of C. pneumoniae and atherosclerosis by confirming the presence of viable bacteria within atherosclerotic plaque.