Total knee arthroplasty patients displaying symptoms consistent with either implant biomechanical failure or periprosthetic infection, and requiring revision surgery, had synovial fluid aspirated from the affected joint and subjected to standard microbiological culture testing for bacterial infection. In this study, an aliquot of each synovial fluid specimen was analyzed for infection using the rapid, sensitive molecular assay of polymerase chain reaction (PCR), designed to selectively amplify bacterial deoxyribonucleic acid. Decisions concerning surgical procedures performed during revision surgery were based entirely on clinical criteria, including standard preoperative evaluation for infection, independent of PCR results. In total, on the basis of pre- and/or intraoperative clinical data in conjunction with postoperative information, septic failure was determined in 31 of 50 patients, all of whom tested positive using the PCR assay. This correlative study validates the application of the PCR assay for clinical evaluation of arthroplasty infections.