Matrix-associated autologous chondrocyte transplantation/implantation (MACT/MACI) is a new operation procedure using a cell seeded collagen matrix for the treatment of localized full-thickness cartilage defects. A prospective clinical investigation was carried out in order to clarify whether this proves suitable and confirms objective and subjective clinical improvement over a period of up to 5 years after operation. Thirty-eight patients with localised cartilage defects were treated with MACT. Within the context of clinical follow-up, these patients were evaluated for up to 5 years after the intervention. Four different scores (Meyers score, Tegner-Lysholm activity score, Lysholm-Gillquist score, ICRS score) as well as the results of six arthroscopies and biopsies obtained from four patients formed the basis of this study. For 15 patients, 5 or more years had elapsed since the operation at the time this study was completed. It was possible to obtain results 5 years postoperatively from 11 (73.3%) of these 15 patients. Overall, we included 25 patients into the evaluation with a 2-year or longer postoperative period. Five years after transplantation 8 out of 11 patients rated the function of their knee as much better or better than before. Three of the four scores showed significant improvement compared to the preoperative value. One score, the Tegner-Lysholm score showed improvement, which, however, did not prove to be significant. The significantly improved results on three scores after 5 years suggest that MACT represents a suitable but cost-intensive alternative in the treatment of local cartilage defects in the knee.