Prophylactic antibiotics are frequently withheld until cultures are obtained in revision total knee arthroplasty (TKA). We undertook a prospective study to determine whether prophylactic preoperative intravenous antibiotics would affect the results of cultures obtained intraoperatively. We enrolled 25 patients with 26 infected TKAs, a known preoperative infecting organism, and no recent antibiotic therapy. Reaspiration of the infected TKA was performed after anesthesia and sterile preparation. Intravenous antibiotic prophylaxis was then administered and the tourniquet inflated. Intraoperative culture swabs and tissue were obtained at arthrotomy. The timing of events was recorded. Pre- and postantibiotic culture data were analyzed to determine the effect of intravenous preoperative prophylactic antibiotics on cultures obtained intraoperatively. Infections were acute postoperative (four), chronic (19), and acute hematogenous (three). The most common infecting organism was cloxacillin-sensitive Staphylococcus aureus (nine knees [35%]). Preoperative prophylactic antibiotics did not affect the results of intraoperative cultures and we therefore believe should not be withheld before surgery for an infected TKA when an organism has been identified on aspiration preoperatively, and there has been no recent (4 weeks) antimicrobial therapy. Level of Evidence: Level II, diagnostic study. See Guidelines for Authors for a complete description of levels of evidence.