Purpose: Degenerative cartilage lesions present a negative joint environment, which may have a negative effect on the process of cartilage regeneration. The aim of this study is to analyze the clinical outcome obtained with the treatment for isolated degenerative knee cartilage lesions by second-generation arthroscopic autologous chondrocyte implantation (ACI). Methods: Fifty-eight consecutive patients affected by focal degenerative chondral lesions of the femoral condyles and trochlea were treated by second-generation arthroscopic ACI. The mean age at surgery was 34. 7 ± 9. 1 years and the average defect size was 2. 3 ± 0. 9 cm2. The patients were prospectively evaluated with IKDC, EQ-VAS, and Tegner scores preoperatively, at 2 and 6 years. Results: A statistically significant improvement was observed in all scores from the basal evaluation to the final follow-up. The IKDC subjective score improved from 39. 3 ± 13. 6 to 68. 8 ± 22. 7 and 68. 5 ± 23. 9 at the 2- and 6-year follow-ups, respectively, with a significant improvement (P < 0. 0005) and stable results over time; the same trend was confirmed by the EQ-VAS and Tegner scores. The worst results were found in patients with a low physical activity level, women, and those having undergone previous surgery, whereas the symptom duration before surgery did not influence the final outcome. The failure rate was 18. 5%. Conclusions: Despite a significant improvement, the results were lower with respect to the outcome reported in different study populations, and the number of failures was markedly higher, too. Tissue-engineered cartilage implantation is a promising approach for the treatment of degenerative chondral lesions, but graft properties, besides mechanical and biochemical joint environment, have to be improve