Human β-defensin-3 (hBD-3) has been found in synovial fluid and later in periprosthetic tissues in septic joint implant loosening. The aim of the present study was to identify its cellular sources. Tissue samples from 12 patients were analyzed. A fully automatic Leica BOND MAX staining robot was used. Affinity-purified rabbit anti-human hBD-3 IgG was applied in a two-layer horse radish peroxidase/anti-rabbit-labeled polymer method. Double immunofluorescence of hBD3 together with CD68, CD31, heat shock protein 47 (HSP47) and mast cell tryptase (MCT) staining was done. Human BD-3 was found in monocyte/macrophage-like cells, vascular endothelial cells and fibroblasts-like cells, but was weakly expressed in foreign body giant cells and negative in neutrophils. Human BD-3 was found in CD68 and CD31 immunoreactive cells, whereas HSP47 and MCT positive cells were hBD-3 negative. Immunostaining of hBD-3 was strong in some tissue areas but weak or absent in others. Monocyte/macrophages and endothelial cells were established in this study as the major cellular sources of hBD-3 in septic loosening, but fibroblasts and foreign body giant cells can also contribute to its production. The heterogeneous topological staining of hBD-3 suggests local regulation, possibly by bacterial products, damage-associated molecular patterns and cytokines. The results explain the increased synovial fluid/tissue concentrations of hBD-3 in septic loosening.