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Obtaining reliable cultures during revision arthroplasty is important to adequately diagnose and treat a prosthetic joint infection (PJI). The influence of antimicrobial prophylaxis on culture results remains unclear. Since withholding prophylaxis increases the risk for surgical site infections, clarification on this topic is critical. A systematic review was performed with the following research question: in patients who undergo revision surgery of a prosthetic joint, does preoperative antimicrobial prophylaxis affect the culture yield of intraoperative samples in comparison with nonpreoperative antimicrobial prophylaxis? Seven articles were included in the final analysis. In most studies, standard diagnostic culture techniques were used. In patients with a PJI, pooled analysis showed a culture yield of 88% (145/165) in the prophylaxis group versus 95% (344/362) in the nonprophylaxis group (P = 0.004). Subanalysis of patients with chronic PJIs showed positive cultures in 88% (78/89) versus 91% (52/57), respectively (P = 0.59). In patients with a suspected chronic infection, a maximum difference of 4% in culture yield between the prophylaxis and nonprophylaxis groups was observed. With the use of standard culture techniques, antimicrobial prophylaxis seems to affect cultures in a minority of patients. Along with the known risk of surgical site infections due to inadequate timing of antimicrobial prophylaxis, we discourage the postponement of prophylaxis until tissue samples are obtained in revision surgery. Future studies are necessary to conclude whether the small percentage of false-negative cultures after prophylaxis can be further reduced with the use of more-sensitive culture techniques, like sonication.