Introduction <p>Two-stage revision is considered the gold standard treatment in chronic periprosthetic joint infection (PJI) but no specific criteria or examination exist to determine infection eradication before reimplantation. This study aimed to assess the diagnostic performance of leukocyte scintigraphy after the first-stage procedure in two-stage revision for chronic PJI.</p> Material and methods <p>Patients studied with leukocyte scintigraphy after cement spacer insertion for knee PJI from January 2012 to December 2021 were retrospectively included. Infection was diagnosed using the criteria of the 2018 International Consensus Meeting. When 1 or more minor criteria were positive but the score was &lt; 6, patients were considered not infected if there was no recurrence of infection at least 24 months after the second-stage procedure. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were found.</p> Results <p>The final cohort included 67 cases in 61 patients (M: F = 32:35; mean age 74 years). There were 43 true negatives, 8 true positives, 12 false negatives, and 4 false positives. The sensitivity and specificity of leukocyte scintigraphy in diagnosing PJI were respectively 40.0% and 91.5%, PPV was 66.7%, NPV was 78.2%, and accuracy was 76.1%. Staphylococcus Epidermidis was the most frequently isolated microorganism (50%).</p> Conclusion <p>Due to the high costs and the difficulty in its execution, LLS should not be used routinely but it could represent an additional criterion in doubtful cases. In any case, a thorough evaluation of other pre- and intra-operative tests is essential to determine whether reimplantation or spacer renewal is the best course of action. Special consideration should be given to positive LLS, as its results are highly specific and have a low rate of false positives. Conversely, in over half of infected patients, LLS could give false negatives, making negative LLS results less relevant.</p>