In this prospective study we present a novel technique of matrix-based chondrocyte transplantation for the treatment of focal cartilage defects. 22 patients with chondral or osteochondral femoral defects were treated with 3D-collagen type-I gel seeded with autologous chondrocytes (CaReS). Overall handling of this novel method is described, and results after 2 years follow-up with regard to the ICRS-score are presented. 14 patients were treated because of a focal chondral lesion, in 8 cases subchondral bone had to be reestablished. The average defect size was 6 cm(2). In all cases cell-seeded matrix samples could be fixed with fibrin glue without any problems. The average surgery time was 69 min, length of the surgical approach was 8.2 cm. No surgery-specific problems had to be observed. A significant improvement in IKDC-score, functional score and overall rating after 3 or 6 months up to 2 years follow-up was detected. After 2 years post-OP 13 Patients were evaluated and matrix-based defect treatment by CaReS was rated good to excellent by 84.6 % of the patients. Defect treatment with cell-seeded 3D-collagen gel is easy to handle with a good clinical outcome after 2 years follow-up. Further investigations with higher patient number, longer follow-up time and histological and biomechanical sample analysis are needed in order to establish this method as an improvement of conventional ACT.