Osteochondral defects of the ankle are common lesions affecting the talar cartilage and subchondral bone. Current treatments include cell-based therapies but are frequently associated with donor-site morbidity. Our objective is to characterize the posterior process of the talus (SP) and the os trigonum (OT) tissues and investigate its potential as a new source of viable cells for application in tissue engineering and regenerative medicine.SP and OT tissues obtained from six patients were characterized by micro-computed tomography, and histological, histomorphometric and immunohistochemical analyses. Isolated cells proliferation and viability were evaluated by MTS assay, DNA quantification and Live/Dead staining. The TUNEL assay was performed to evaluate cell death by apoptosis. Moreover, the production of extracellular matrix was evaluated by toluidine blue staining, whereas cells phenotype was investigated by flow cytometry. Ankle explants characterization showed the presence of a cartilage tissue layer in both SP and OT tissues, which represent, at least 20% in average of the explant. The presence of type II collagen was detected in the extracellular matrix. Isolated cells presented a round morphology typical of chondrocytes. In in vitro studies, cells were viable and proliferating up to 21 days of culturing. No signs of apoptosis were detected. Flow cytometry analysis revealed that isolated cells maintained the expression of several chondrocytic markers during culturing. The results indicate that the SP and OT tissues are a reliable source of viable chondrocytes, which can find promising applications in ACI/MACI strategies with minimal concerns regarding donor zone complications.