Two-year follow-up to assess efficacy and safety of high-density autologous chondrocyte implantation (HD-ACI) in patients with cartilage lesions in the ankle. Twenty-four consecutive patients with International Cartilage repair Society (ICRS) grade 3-4 cartilage lesions of the ankle were included. Five million chondrocytes per cm2 of lesion were implanted using a type I/III collagen membrane as a carrier and treatment effectiveness was assessed by evaluating pain with the visual analogue scale (VAS) and American Orthopaedic Foot & Ankle Society (AOFAS) ankle-hindfoot score at baseline, 12-month, and 24-month follow-up, together with dorsal and plantar flexion. Magnetic resonance observation for cartilage repair tissue (MOCART) score was used to evaluate cartilage healing. Histological study was possible in 5 cases. Patients' median age was 31 years (range 18-55 years). Median VAS score was 8 (range 5-10) at baseline, 1.5 (range 0-8) at 12-month follow-up, and 2 (rang e0-5) at 24-month follow-up (P < 0.001). Median AOFAS score was 39.5 (range 29-48) at baseline, 90 (range 38-100) at 12-month follow-up, and 90 (range 40-100) at 24-month follow-up (P < 0.001). Complete dorsal flexion significantly increased at 12 months (16/24, 66.7%) and 24 months (17/24, 70.8%) with regard to baseline (13/24, 54.2%) (P = 0.002). MOCART at 12- and 24-month follow-ups were 73.71 ± 15.99 and 72.33 ± 16.21. Histological study confirmed that neosynthetized tissue was cartilage with hyaline extracellular matrix and numerous viable chondrocytes. HD-ACI is a safe and effective technique to treat osteochondral lesions in the talus, providing good clinical and histological results at short- and mid-term follow-ups.