We analysed the improvement of cartilage defect regeneration by the use of microspheres of autologous chondrocytes. Autologous chondrocytes from minipigs were cultured using the microsphere technology. Cartilage defects were surgically introduced to tibia-femoral joints. Tissue constructs were then implanted into defect sites. Histological, immunohistological and transmission electron microscopic analyses were performed. Histological and ultrastructural investigations of chondrospheres revealed a cartilage-like tissue formation, indicated by phenotypically differentiated chondrocyte-like cells surrounded by de novo synthesised chondrogenic extracellular matrix. Clinical inspection of defects demonstrated nearly complete cartilage regeneration in the microtissue treated defect sites, whereas controls exhibited irregular fibrous tissue formation. In vitro-expanded articular chondrocytes are able to form a microtissue capable of repairing cartilage defects in vivo, improving regeneration of cartilage defects.