Purpose Unexpected-positive-intraoperative-cultures (UPIC) are common in presumed aseptic revision-total-knee-arthroplasties (rTKA). However, the clinical signiicance is not entirely clear. In contrast, in some presumably septic rTKA, identiication of an underlying pathogen was not possible, so-called unexpected-negative-intraoperative-cultures (UNIC). The purpose of this study was to evaluate the potential use of synovial alpha-defensin (AD) levels in these patients. Methods Synovial AD levels from 143 rTKAs were evaluated retrospectively from our prospectively maintained institutional periprostetic joint infection (PJI) biobank and database. The 2018-International Consensus Meeting (ICM) criteria was used to deine the study groups. Samples from UPICs with a minimum of one positive intraoperative culture (ICM 2-≥ 6) (n = 20) and UNIC's (ICM ≥ 6) (n = 14) were compared to 34 septic culture-positive samples (ICM ≥ 6) and 75 aseptic culture-negative (ICM 0-1). Moreover, AD-lateral-low-assay (ADLF) and an enzyme-linked-immunosorbent-assay (ELISA) in detecting the presence of AD in native and centrifuged synovial luid specimens was performed. Concentration of AD determined by ELISA and ADLF methods, as well as microbiological, and histopathological results, serum and synovial parameters along with demographic factors were analysed. Results AD was positive in 31/34 (91.2%) samples from the septic culture-positive group and in 14/14 (100%) samples in the UNIC group. All UPIC samples showed a negative AD result. Positive AD samples were highly associated with culture positive and histopathological results (p < 0.001). No high-virulent microorganisms (0/20) were present in the UPIC group, compared to infected-group (19/34; 55.9%). High virulent microorganisms showed a positive AD result in 89.5% (17/19) of the cases. Methicillin resistant Staphylococcus epidermis (MRSE) infections had signiicantly higher AD levels than with methicillin susceptible S. epidermdis (MSSE) (p = 0.003). ELISA and ADLF tests were positive with centrifuged (8/8) and native (8/8) synovial luid. Conclusion AD showed a solid diagnostic performance in infected and non-infected revisions, and it provided an additional value in the diagnosis of UPIC and UNIC associated to rTKAs. Pathogen virulence as well as antibiotic resistance pattern may have an efect on AD levels. Centrifugation of synovial luid had no inluence on ADLF results.