Through the use of polymerase chain reaction (PCR)-electron spray ionization (ESI)-time of flight (TOF)-mass spectrometry (MS), we identified multiple periodontal pathogens within joint tissues of individuals undergoing replacement arthroplasties of the knee. The most prevalent of the periodontal pathogens were Treponema denticola and Enterococcus faecalis, the latter of which is commonly associated with apical periodontitis. These findings were unique to periprosthetic joint infections (PJI) of the knee and were never observed for PJIs of other lower extremity joints (hip and ankle) or upper extremity joints (shoulder and elbow). These data were confirmed by multiple independent methodologies including fluorescent in situ hybridization (FISH) which showed the bacteria deeply penetrated inside the diseased tissues, and 454-based deep 16S rDNA sequencing. The site-specificity, the tissue investment, and the identical findings by multiple nucleic-acid-based techniques strongly suggests the presence of infecting bacteria within these diseased anatomic sites. Subsequently, as part of a control program using PCR-ESI-TOF-MS, we again detected these same periodontal pathogens in aspirates from patients with osteoarthritis who were undergoing primary arthroplasty of the knee and thus who had no history of orthopedic implants. This latter finding raises the question of whether hematogenic spread of periodontal pathogens to the knee play a primary or secondary-exacerbatory role in osteoarthritis.