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Treating articular cartilage defects is a demanding problem. Although several studies have reported durable and improved clinical outcomes after autologous chondrocyte implantation (ACI) over a long-term period, there is no report with over 20 years’ follow-up.To evaluate clinical outcomes after first-generation ACI for the treatment of knees with disabling, large single and multiple cartilage defects for which patients wished to avoid prosthetic arthroplasty, with a minimum of 20 years’ follow-up.Case series; Level of evidence, 4.The authors reviewed prospectively collected data from 23 patients (24 knees; mean age, 35.4 years [range, 13-52 years]) undergoing ACI for the treatment of symptomatic, full-thickness articular cartilage lesions. A mean of 2.1 lesions per knee were treated over a mean total surface area of 11.8 cm2 (range, 2.4-30.5 cm2) per knee. Kaplan-Meier survival analysis and functional outcome scores, including the modified Cincinnati Knee Rating System, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Short Form–36 (SF-36), were used. Patients also self-reported an improvement in pain with a visual analog scale and a satisfaction survey.The 20-year survival rate was 63% (95% CI, 40%-78%). The evaluation of the 15 knees with retained grafts demonstrated that all clinical scores except the WOMAC subscore for stiffness and SF-36 mental component summary score improved significantly and were sustained to 20 years postoperatively. Ninety-three percent of these patients rated knee-specific outcomes as good or excellent. The outcomes for 9 of 24 knees were considered failures, including 5 undergoing revision ACI and 4 being converted to arthroplasty at a mean of 1.7 and 5.9 years, respectively. Only 1 of 5 knees that underwent revision ACI was converted to arthroplasty at 1.9 years after the index surgery, and the other 4 patients were able to maintain their biological knee. Overall, 20 years later, 79% of patients maintained their native knee, for which they initially sought treatment, and were satisfied when evaluated.First-generation ACI provided satisfactory survival rates and significant clinical improvements over a 20-year follow-up, which offers an important standard for comparison with newer-generation ACI technologies of the future.