We reported our experience with the usefulness and interpretation of the significance of cultures at the second-stage exchange of infected arthroplasty in a prospective, 3-year follow-up study. When such intraoperative cultures were negative, patients received no therapy; when at least two cultures showed the same microorganism, results were interpreted as infection and patients were treated with antibiotics for 6-8 weeks with no more surgical procedures. Genotypic analysis (pulsed-field gel electrophoresis) was performed to analyse coagulase-negative Staphylococcus (CoNS) infections. Among 25 patients, 18 had negative cultures at the second-stage (group 1) and seven had positive cultures (group 2) receiving glycopeptides. Follow-up medians were 30 months for group 1 and 35 months for group 2; no patients in either group had persistence or recurrence of infection. All patients from group 2 had infection by CoNS at the second-stage; in six cases CoNS were also responsible for the initial infection. Genetic studies confirm that second-stage strains show different clonal identity than first-stage ones suggesting a superinfection rather than a real persistence of initial infection. Our results support the role of intraoperative cultures at the second-stage to identify patients at risk of recurrent infection who could benefit from early antibiotic therapy. The persistence of initial infections and the presence of new superinfections should be better defined according to genotypic studies.