Microfracture is a frequently used reparative technique that induces a healing response in articular cartilage defects. Penetration of the subchondral bone leads to blood clot formation, allows multipotent mesenchymal cells to access the defect and, subsequently, leads to cartilaginous repair tissue. The aim of our study was to analyze the chemotactic recruitment of human subchondral spongious bone marrow-derived cells by synovial fluid (SF) from normal donors (ND), patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Subchondral spongious bone marrow-derived mesenchymal progenitors were isolated from bone cylinders after high tibial osteotomy and analyzed for the presence of stem cell-related cell surface antigens by flow cytometry. Recruitment of subchondral progenitors by normal SF and SF from donors with degenerated joint diseases was documented by using a modified Boyden chamber chemotaxis assay. The chemotaxis assay demonstrated that synovial fluid has the potential to recruit mesenchymal progenitors in vitro. SF from normal donors and patients with OA showed no difference in the potential to stimulate cell migration. SF obtained from RA donors showed significantly reduced cell recruitment compared to SF derived from OA patients (p = 0.0054) and normal donors (p < 0.0001). The chemotactic activity of SF obtained from normal donors and from patients with degenerative joint diseases suggests that SF may be actively involved in the migration of progenitors in cartilage defects after microfracture.